Real-time pcr for the detection of pathogens

ABSTRACT

Methods for detecting presence of one or more of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumooniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcus agalactiae, and Neisseria meningitidis nucleic acids in a sample, such as a biological sample obtained from a subject, or an environmental sample, are provided. This disclosure also provides probes, primers, and kits for detecting one or more of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcus agalactiae, and Neisseria meningitidis in a sample.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of co-pending U.S. application Ser. No. 16/056,147, filed Aug. 6, 2018, which is a divisional of U.S. application Ser. No. 14/398,390, filed Oct. 31, 2014, now U.S. Pat. No. 10,072,305, issued Sep. 11, 2018, which is the § 371 U.S. National Stage of International Application No. PCT/US2013/028034, filed Feb. 27, 2013, which was published in English under PCT Article 21(2), which in turn claims the benefit of U.S. Provisional Application No. 61/642,091, filed May 3, 2012, each of which is incorporated herein by reference in its entirety.

FIELD

This disclosure concerns methods and compositions related to the detection of pathogens, particularly utilizing real-time PCR.

BACKGROUND

Many pathogens have major public health and economic impact. Pathogens may be spread in the community or in a clinic or hospital setting, and multidrug resistance is a growing problem in many pathogens. Furthermore, although a presumptive clinical diagnosis can often be made through symptomology, a laboratory identification determining the etiology of a disease is critical to establish the correct course of treatment. Current tests for many pathogens are neither highly sensitive nor specific, and in some cases require an acute and convalescent patient serum (paired serum) for clear identification. Thus, a need remains for rapid, cost-effective, sensitive, and specific assays for many pathogens. In particular, there is a need for assays for diagnosing and differentiating major pathogens of childhood and neonatal infection, which cause significant neonatal mortality throughout the world.

SUMMARY

Disclosed herein are methods for detecting presence of one or more of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcus agalactiae (Group B Streptococcus; GBS), and/or Neisseria meningitidis nucleic acids in a sample, such as a biological sample obtained from a subject, or an environmental sample. In particular, these pathogens are some of the most common causes of infection in neonates and young children, and are major causes of mortality in children under the age of five. The methods provided herein include simultaneous multipathogen detection assays, which can be used to diagnose and differentiate causes of neonatal infection. In addition, since all of these assays target infectious agents, all population groups may benefit from the advancements described herein. Depending on the specific circumstances and epidemiological data associated with sporadic and/or outbreak-linked cases, the disclosed methods can provide a valuable diagnostic tool for the clinician and medical epidemiologist charged with determining the etiology of a disease.

The disclosed methods can be used to detect presence of one or more (or any combination of two or more thereof) of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcus agalactiae, and/or Neisseria meningitidis nucleic acids in a sample, for example, by contacting a sample with one or more of the probes disclosed herein (such as one or more of SEQ ID NOs: 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 56, 60, and 64) and detecting hybridization of one or more of the probes with a nucleic acid in the sample. The disclosed methods provide rapid, sensitive, and specific detection of these organisms, for example, utilizing real-time simultaneous multipathogen detection or a multiplex real-time PCR assay.

In some embodiments, the disclosed methods further include amplifying one or more (or any combination of two or more thereof) of an Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcus agalactiae, and/or Neisseria meningitidis nucleic acid, for example utilizing one or more primers (such as one or more of SEQ ID NOs: 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28, 30, 31, 33, 34, 36, 37, 39, 40, 42, 43, 45, 46, 48, 49, 51, 54, 55, 58, 59, 62, and/or 63).

This disclosure also provides kits for detecting one or more of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumooniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcus agalactiae, and/or Neisseria meningitidis in a sample, for example, including one or more of the probes and primers disclosed herein.

The foregoing and other features, of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are a series of graphs showing the effect of lytic enzyme treatment on extraction of nucleic acid from blood specimens. Average Ct value of individual real-time PCR reactions (n=4) containing TNA extracted from healthy donor blood spiked with serial dilutions of S. aureus (FIG. 1A) or K. pneumoniae (FIG. 1B) without treatment, after incubation with TE buffer alone, or after treatment with TE buffer with lytic enzymes (lysozyme, lysostaphin, and mutanolysin) at 37° C. for 30 minutes. FIG. 1C shows Ct values of serial dilutions of K. pneumoniae spiked into saline (to mimic NP/OP swab) or blood. Error bars display standard deviation. * p<0.0001 compared to no treatment. #p<0.05 compared to same concentration of organisms in saline.

FIGS. 2A-2B are a pair of graphs showing concordance between replicates of primary clinical specimens tested on a TAQMAN® array card (TAC). Concordance between replicate results for nasopharyngeal/oropharyngeal (NP/OP) (FIG. 2A) and blood (FIG. 2B) specimens tested using TAC. Data shown are total number of specimens identified as positive in at least one replicate reaction (white bars) and proportion of positive specimens for which greater than 50% of replicates were positive (shaded bars). Number of replicates tested varied by target and specimen type; all targets were tested in ≥2 replicates. Total number of specimens tested, NP/OP (n=124), blood (n=661). ADEV, Adenovirus; BOP1, Bordetella pertussis; ENTV, Enterovirus; FLUA, Influenza A; GBST, Group B Streptococcus; HMPV, Human Metapneumovirus; RESV, Respiratory Syncytial Virus; RHIV, Rhinovirus; STPN, Streptococcus pneumoniae; URUP, Ureaplasma spp.; CYMV, Cytomegalovirus; ECSH, Escherichia coli/Shigella spp.; GAST, Group A Streptococcus; HIAT; Haemophilus influenzae; KLPN, Klebsiella pneumoniae, PSAE, Pseudomonas aeruginosa, SALS, Salmonella spp.; STAU, Staphylococcus aureus.

FIGS. 3A and 3B are a pair of diagrams showing effect of enzyme system on detection of pathogen targets in primary clinical specimens. Data shown are difference in Ct value between reactions using Quanta One-step RT-PCR TOUGHMIX® and AGPATH-ID™ One-step RT-PCR kit when testing TNA extracted from NP/OP swabs (FIG. 3A) or blood (FIG. 3B). Each data point represents the difference in Ct value between the two reactions for an individual clinical specimen. Median difference is indicated (−) for assays with ≥2 positive results. *Targets that were only detected using AGPATH-ID™ always occurred when Ct values were >33.

SEQUENCE LISTING

Any nucleic acid and amino acid sequences listed herein or in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. § 1.822. In at least some cases, only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.

The Sequence Listing is submitted as an ASCII text file in the form of the file named Sequence_Listing.txt, which was created on Sep. 27, 2021, and is 41,259 bytes, which is incorporated by reference herein.

SEQ ID NO: 1 is an exemplary Acinetobacter baumannii oxa-51 nucleic acid sequence.

SEQ ID NO: 2 is an exemplary Chlamydia trachomatis tmRNA nucleic acid sequence.

SEQ ID NO: 3 is an exemplary Escherichia coli uidA nucleic acid sequence.

SEQ ID NO: 4 is an exemplary Klebsiella pneumoniae nifA nucleic acid sequence.

SEQ ID NO: 5 is an exemplary Moraxella catarrhalis purH nucleic acid sequence.

SEQ ID NO: 6 is an exemplary Pneumocystis jirovecii dhps nucleic acid sequence.

SEQ ID NO: 7 is an exemplary Pseudomonas aeruginosa gyrB nucleic acid sequence.

SEQ ID NO: 8 is an exemplary Staphylococcus aureus gsf nucleic acid sequence.

SEQ ID NO: 9 is an exemplary Toxoplasma gondii ssrRNA nucleic acid sequence.

SEQ ID NO: 10 is an exemplary Ureaplasma parvum ure nucleic acid sequence.

SEQ ID NO: 11 is an exemplary Ureaplasma urealyticum mba nucleic acid sequence.

SEQ ID NOs: 12-14 are exemplary Acinetobacter baumannii oxa-51 primer and probe nucleic acid sequences.

SEQ ID NOs: 15-17 are exemplary Pseudomonas aeruginosa gyrB primer and probe nucleic acid sequences.

SEQ ID NOs: 18-20 are exemplary Klebsiella pneumoniae nifA primer and probe nucleic acid sequences.

SEQ ID NOs: 21-23 are exemplary Toxoplasma gondii ssrRNA primer and probe nucleic acid sequences.

SEQ ID NOs: 24-26 are exemplary Moraxella catarrhalis purH primer and probe nucleic acid sequences.

SEQ ID NOs: 27-29 are exemplary Escherichia coli/Shigella spp. primer and probe nucleic acid sequences.

SEQ ID NOs: 30-32 are exemplary Staphylococcus aureus gsf primer and probe nucleic acid sequences.

SEQ ID NOs: 33-35 are exemplary Pneumocystis jirovecii dhps primer and probe nucleic acid sequences.

SEQ ID NOs: 36-38 are exemplary Chlamydia trachomatis tmRNA primer and probe nucleic acid sequences.

SEQ ID NOs: 39-41 are exemplary Ureaplasma urealyticum mba primer and probe nucleic acid sequences.

SEQ ID NOs: 42-44 are exemplary Ureaplasma parvum ure primer and probe nucleic acid sequences.

SEQ ID NOs: 45-47 are exemplary Ureaplasma spp. ure primer and probe nucleic acid sequences.

SEQ ID NOs: 48-51 are exemplary Bartonella spp. ssrA primer and probe nucleic acid sequences.

SEQ ID NO: 52 is an exemplary Bartonella ssrA nucleic acid sequence.

SEQ ID NO: 53 is an exemplary Group B Streptococcus cfb nucleic acid sequence.

SEQ ID NOs: 54-56 are exemplary Group Streptococcus cfb primer and probe nucleic acid sequences.

SEQ ID NO: 57 is an exemplary Klebsiella pneumoniae diguanylate cyclase nucleic acid sequence.

SEQ ID NOs: 58-60 are exemplary Klebsiella pneumoniae diguanylate cyclase primer and probe nucleic acid sequences.

SEQ ID NO: 61 is an exemplary Neisseria meningitidis sodC nucleic acid sequence.

SEQ ID NOs: 62-64 are exemplary Neisseria meningitidis sodC primer and probe nucleic acid sequences.

SEQ ID NOs: 65-67 are exemplary Salmonella spp. ttrRsBCA primer and probe nucleic acid sequences.

DETAILED DESCRIPTION I. Abbreviations

BAL: bronchoalveolar lavage

GBS: Group B Streptococcus

NP: nasopharyngeal

NTC: no template control

OP: oropharyngeal

TAC: TAQMAN array card

TNA: total nucleic acids

UTM: universal transport medium

II. Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. As used herein, “comprises” means “includes.” Thus, “comprising A or B,” means “including A, B, or A and B,” without excluding additional elements. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes.

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:

Acinetobacter baumannii: An aerobic gram-negative bacterium which can cause pneumonia, urinary tract infection, and necrotizing fasciitis. Many strains of A. baumannii are antibiotic resistant and it is an increasingly common nosocomial infection, for example, in intensive care units (such as neonatal intensive care units). A. baumannii can also colonize solutions (such as irrigating or intravenous solutions). Nucleic acid and protein sequences for A. baumannii are publicly available. For example, GenBank Accession Nos. NC_011586, NC_011595, NC_010611, NC_009085, and NC_010410 provide exemplary A. baumannii genome sequences, all of which are incorporated by reference as provided by GenBank on Apr. 30, 2012.

The A. baumannii oxa-51 gene encodes a beta-lactamase. Exemplary A. baumannii oxa-51 nucleic acid sequences include GenBank Accession Nos. AJ309734 and DQ385606, both of which are incorporated by reference as present in GenBank on Apr. 30, 2012. An exemplary Acinetobacter baumannii nucleotide sequence of oxa-51 is found at GenBank Accession No. AJ309734 (SEQ ID NO: 1).

Bartonella spp.: A genus of gram-negative bacteria that infect a wide variety of mammalian hosts, including humans. Bartonella are transmitted by blood-sucking insects (for example, ticks, fleas, and lice). High prevalence of Bartonella bacteremia has been reported in populations of rodents, cats, and ruminants worldwide. There are at least 30 known species and subspecies of Bartonella. Nucleic acid and protein sequences for Bartonella spp. are publicly available. For example, GenBank Accession Nos. NC_005955 (B. quintana), NC_005956 (B. henselae), NC_010161 (B. tribocorum), NC_012846 (B. grahamii), and NC_008783 (B. bacilliformis) provide exemplary Bartonella genome sequences, all of which are incorporated by reference as provided by GenBank on Apr. 30, 2012. One of ordinary skill in the art can identify additional Bartonella spp., for example utilizing the NCBI Taxonomy Browser (e.g., www.ncbi.nlm.nih.gov/Taxonomy/).

The Bartonella ssrA RNA (also known as transfer-messenger RNA; tmRNA) is a single-copy prokaryotic-specific molecule involved in processing of incomplete peptides and resolution of stalled ribosomes during translation. Exemplary Bartonella ssrA nucleic acid sequences include GenBank Accession Nos. JN029766, BX897700 (1020848 . . . 1021176), JN029785, BX87699 (1215947 . . . 1216279), JN029796, AM260525 (1675346 . . . 1675682), JN029795, NC_012846.1 (1542284 . . . 1542620), JN029794, and NC_008783.1 (955069 . . . 955373), all of which are incorporated herein by reference as provided by GenBank on Apr. 30, 2012. An exemplary Bartonella nucleotide sequence of ssrA is found at GenBank Accession No. NC_005955 (1020727-1021342) (SEQ ID NO: 52).

Chlamydia trachomatis: A gram-negative bacterium that is a common sexually transmitted disease. C. trachomatis can also be transmitted from an infected mother, resulting in a potentially life-threatening respiratory infection in neonates. Nucleic acid and protein sequences for C. trachomatis are publicly available. For example, GenBank Accession Nos. NC_012687, NC_000117, NC_007429, NC_010280, and NC_015744 provide exemplary C. trachomatis genome sequences, all of which are incorporated by reference as provided by GenBank on Apr. 30, 2012.

The C. trachomatis tmRNA is an RNA with both tRNA and mRNA characteristics. Exemplary C. trachomatis tmRNA nucleic acid sequences include GenBank Accession Nos. NC_000117 (20663-21082) and NC_007429 (21258-21677; complement), both of which are incorporated herein by reference as present in GenBank on Apr. 30, 2012. An exemplary Chlamydia trachomatis nucleotide sequence of tmRNA is found at GenBank Accession No. NC_000117 (20663-21082) (SEQ ID NO: 2).

Escherichia coli: A Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms, where it (and related bacteria) constitute about 0.1% of gut flora. Most E. coli strains are harmless, but some serotypes (for example O157:H7 and O104:H4) can cause serious disease in humans. Pathogenic E. coli is frequently contracted via contaminated food or water. The harmless strains are part of the normal flora of the gut, and can benefit their hosts by producing vitamin K₂ and by preventing the establishment of pathogenic bacteria within the intestine.

Nucleic acid and protein sequences for E. coli are publicly available. For example, GenBank Accession Nos. NC_011751, NC_011742, NC_011415, NC_011601, and NC_011353 provide exemplary E. coli genome sequences, all of which are incorporated by reference as provided by GenBank on Apr. 30, 2012.

The E. coli uidA gene encodes a β-D-glucuronidase. Exemplary E. coli uidA nucleic acid sequences include GenBank Accession Nos. NC_011601 (1769353-1771164, complement), NC_000913 (1692284-1694095), and NC_011751 (192037-1930848, complement), all of which are incorporated herein by reference as present in GenBank on Apr. 30, 2012. An exemplary E. coli nucleotide sequence of uidA is found at GenBank Accession No. NC_000913 (1692284-1694095) (SEQ ID NO: 3).

Fluorophore: A chemical compound, which when excited by exposure to a particular stimulus, such as a defined wavelength of light, emits light (fluoresces), for example at a different wavelength (such as a longer wavelength of light).

Fluorophores are part of the larger class of luminescent compounds. Luminescent compounds include chemiluminescent molecules, which do not require a particular wavelength of light to luminesce, but rather use a chemical source of energy. Therefore, the use of chemiluminescent molecules (such as aequorin) eliminates the need for an external source of electromagnetic radiation, such as a laser.

Examples of particular fluorophores that can be used in the probes and primers disclosed herein are known to those of ordinary skill in the art and include those provided in U.S. Pat. No. 5,866,366 to Nazarenko et al., such as 4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-1-naphthyl)maleimide, anthranilamide; Brilliant Yellow; coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI); 5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid; 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid; 5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride); 4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), QFITC (XRITC), 6-carboxy-fluorescein (HEX), and TET (tetramethyl fluorescein); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4-methylumbelliferone; ortho-cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate, and succinimidyl 1-pyrene butyrate; Reactive Red 4 (CIBACRON™ Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethyl rhodamine, and tetramethyl rhodamine isothiocyanate (TRITC); sulforhodamine B; sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); riboflavin; rosolic acid and terbium chelate derivatives; LightCycler Red 640; Cy5.5; and Cy56-carboxyfluorescein; boron dipyrromethene difluoride (BODIPY); acridine; stilbene; Cy3; Cy5, VIC® (Applied Biosystems); LC Red 640; LC Red 705; and Yakima yellow amongst others. Additional examples of fluorophores include Quasar® 670, Quasar® 570, CAL Fluor® Red 590, CAL Fluor® Red 610, CAL Fluor® 615, CAL Fluor® Red 635, CAL Fluor® Green 520, CAL Fluor® Gold 540, and CAL Fluor® Orange 560 (Biosearch Technologies, Novato, Calif.).

Other suitable fluorophores include those known to those of ordinary skill in the art, for example those available from Molecular Probes/Life Technologies (Carlsbad, Calif.). In particular examples, a fluorophore is used as a donor fluorophore or as an acceptor fluorophore.

“Acceptor fluorophores” are fluorophores which absorb energy from a donor fluorophore, for example in the range of about 400 to 900 nm (such as in the range of about 500 to 800 nm). Acceptor fluorophores generally absorb light at a wavelength which is usually at least 10 nm higher (such as at least 20 nm higher) than the maximum absorbance wavelength of the donor fluorophore, and have a fluorescence emission maximum at a wavelength ranging from about 400 to 900 nm. Acceptor fluorophores have an excitation spectrum that overlaps with the emission of the donor fluorophore, such that energy emitted by the donor can excite the acceptor. Ideally, an acceptor fluorophore is capable of being attached to a nucleic acid molecule.

In a particular example, an acceptor fluorophore is a dark quencher, such as Dabcyl, QSY7 (Molecular Probes), QSY33 (Molecular Probes), BLACK HOLE QUENCHERS™ (Biosearch Technologies; such as BHQO, BHQ1, BHQ2, and BHQ3), ECLIPSE™ Dark Quencher (Epoch Biosciences), or IOWA BLACK™ (Integrated DNA Technologies). A quencher can reduce or quench the emission of a donor fluorophore. In such an example, instead of detecting an increase in emission signal from the acceptor fluorophore when in sufficient proximity to the donor fluorophore (or detecting a decrease in emission signal from the acceptor fluorophore when a significant distance from the donor fluorophore), an increase in the emission signal from the donor fluorophore can be detected when the quencher is a significant distance from the donor fluorophore (or a decrease in emission signal from the donor fluorophore when in sufficient proximity to the quencher acceptor fluorophore).

“Donor Fluorophores” are fluorophores or luminescent molecules capable of transferring energy to an acceptor fluorophore, thereby generating a detectable fluorescent signal from the acceptor. Donor fluorophores are generally compounds that absorb in the range of about 300 to 900 nm, for example about 350 to 800 nm. Donor fluorophores have a strong molar absorbance coefficient at the desired excitation wavelength, for example greater than about 10³ M⁻¹ cm⁻¹.

Hybridization: To form base pairs between complementary regions of two strands of DNA, RNA, or between DNA and RNA, thereby forming a duplex molecule. Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (such as the Na⁺ concentration) of the hybridization buffer will determine the stringency of hybridization. Calculations regarding hybridization conditions for attaining particular degrees of stringency are discussed in Sambrook et al., (1989) Molecular Cloning, second edition, Cold Spring Harbor Laboratory, Plainview, N.Y. (chapters 9 and 11). The following is an exemplary set of hybridization conditions and is not limiting:

Very High Stringency (detects sequences that share at least 90% identity)

Hybridization: 5×SSC at 65° C. for 16 hours

Wash twice: 2×SSC at room temperature (RT) for 15 minutes each

Wash twice: 0.5×SSC at 65° C. for 20 minutes each

High Stringency (detects sequences that share at least 80% identity)

Hybridization: 5×-6×SSC at 65° C.-70° C. for 16-20 hours

Wash twice: 2×SSC at RT for 5-20 minutes each

Wash twice: 1×SSC at 55° C.-70° C. for 30 minutes each

Low Stringency (detects sequences that share at least 60% identity)

Hybridization: 6×SSC at RT to 55° C. for 16-20 hours

Wash at least twice: 2×-3×SSC at RT to 55° C. for 20-30 minutes each.

Klebsiella pneumoniae: A gram-negative facultative anaerobic bacterium commonly found in the normal flora of the mouth, skin, and intestine. K. pneumoniae can cause respiratory disease, typically as a result of a colonized subject aspirating oropharyngeal bacteria into the lower respiratory tract. It is one of the most common causes of neonatal sepsis, especially in developing countries. Many strains of K. pneumoniae are antibiotic resistant and it is an increasingly common nosocomial infection. Nucleic acid and protein sequences for K. pneumoniae are publicly available. For example, GenBank Accession Nos. CP000964, NC_011283, NC_009648, and NC_012731 provide exemplary K. pneumoniae genome sequences, all of which are incorporated by reference as provided by GenBank on Apr. 30, 2012. The K. pneumoniae nifA gene encodes a transcriptional activator involved in the regulation of expression of the nif genes. An exemplary K. pneumoniae nifA nucleic acid sequence includes GenBank Accession No. CP000964 (1752865-1754439), which is incorporated herein by reference as present in GenBank on Apr. 30, 2012. An exemplary Klebsiella pneumoniae nucleotide sequence of nifA is found at GenBank Accession No. CP000964 (1752865-1754439) (SEQ ID NO: 4).

The K. pneumoniae diguanylate cyclase gene encodes an enzyme which catalyzes the formation of cyclic di-GMP from GTP. An exemplary K. pneumoniae diguanylate cyclase nucleic acid sequence includes GenBank Accession No. CP000964 (1413543-1415714), which is incorporated herein by reference as present in GenBank on Feb. 15, 2013. An exemplary Klebsiella pneumoniae nucleotide sequence of diguanylate cyclase is found at GenBank Accession No. CP000964 (1413543-1415714) (SEQ ID NO: 57).

Moraxella catarrhalis: A gram-negative bacterium (previously known as Branhamella catarrhalis) which can cause otitis media, respiratory infections, endocarditis, and meningitis, particularly in newborns and young children. Nucleic acid and protein sequences for K. pneumoniae are publicly available. For example, GenBank Accession No. NC_014147 provides an exemplary M. catarrhalis genome sequence, which is incorporated by reference as provided by GenBank on Apr. 30, 2012.

The M. catarrhalis purH gene encodes a bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase. An exemplary M. catarrhalis purH nucleic acid sequence includes GenBank Accession No. NC_014147 (620865-622463), which is incorporated herein by reference as present in GenBank on Apr. 30, 2012. An exemplary Moraxella catarrhalis nucleotide sequence of purH is found at GenBank Accession No. NC_014147 (620865-622463) (SEQ ID NO: 5).

Neisseria meningitidis: A gram-negative bacterium that is a leading cause of septicemia and life-threatening meningitis in children. There are at least five serogroups based on capsular polysaccharides (A, B, C, Y, and W135) Nucleic acid and protein sequences for N. meningitidis are publicly available. For example, GenBank Accession Nos. CP_002419-CP_002424 provide exemplary N. meningitidis genome sequences, all of which are incorporated by reference as provided by GenBank on Feb. 15, 2013.

The Neisseria meningitidis sodC gene encodes a Cu,Zn superoxide dismutase. Exemplary Neisseria meningitidis sodC nucleic acid sequences include GenBank Accession Nos. CP_002423 (862943-493503) and CP_002422 (1411887-1412447), both of which are incorporated by reference herein as provided by GenBank on Feb. 15, 2013. An exemplary N. meningitidis sodC nucleotide sequence is found at GenBank Accession No. CP_002423 (862943-493503) (SEQ ID NO: 61).

Pneumocystis jirovecii: Formerly classified as Pneumocystis carinii. A nonfilamentous fungus that can cause severe pneumonia in immunocompromised patients and neonates. P. jirovecii may also colonize the lungs of healthy individuals without causing disease. Nucleic acid and protein sequences for P. jirovecii are publicly available.

The P. jirovecii dhps gene encodes a diydropteroate synthase. An exemplary P. jirovecii dhps nucleic acid sequence includes GenBank Accession No. AF139132, which is incorporated herein by reference as present in GenBank on Apr. 30, 2012. An exemplary Pneumocystis jirovecii nucleotide sequence of dhps is found at GenBank Accession No. AF139132 (SEQ ID NO: 6).

Pseudomonas aeruginosa: A gram-negative bacterium that is a leading cause of hospital-acquired infections, including bacteremia, burn/wound infections, and severe pneumonia, including in neonatal intensive care units. This organism is also a common cause of community-acquired skin, ear, and eye infections, often associated with swimming in contaminated recreational facilities. Nucleic acid and protein sequences for P. aeruginosa are publicly available. For example, GenBank Accession Nos. NC_011770, NC_009656, NC_002516, and NC_008463 provide exemplary P. aeruginosa genome sequences, all of which are incorporated by reference as provided by GenBank on Apr. 30, 2012.

The P. aeruginosa gyrb gene encodes a dna gyrase subunit B. Exemplary P. aeruginosa gyrb nucleic acid sequences include GenBank Accession Nos. AB005881, NC_002516 (4275-6695), NC_009656 (4274-6694), and NC_008463 (4275-6695), which are incorporated herein by reference as present in GenBank on Apr. 30, 2012. An exemplary Pseudomonas aeruginosa nucleotide sequence of gyrb is found at GenBank Accession No. AB005881 (SEQ ID NO: 7).

Shigella: A genus of Gram-negative, nonspore forming, non-motile, rod-shaped bacteria closely related to Escherichia coli and Salmonella. The causative agent of human shigellosis, Shigella causes disease in primates, but not in other mammals. It can cause infection in neonates as a result of maternal transmission during delivery. During infection, it typically causes dysentery. Phylogenetic studies indicate that Shigella may be more appropriately treated as subgenus of Escherichia.

Staphylococcus aureus: A gram-positive bacterium which can cause diseases including skin infection, respiratory infection, meningitis, endocarditis, toxic shock syndrome, and sepsis. It is one of the most common nosocomial infections and is increasing in frequency in neonatal intensive care units. Multi-drug resistant strains (including methicillin-resistant S. aureus; MRSA) are increasingly common. Nucleic acid and protein sequences for S. aureus are publicly available. For example, GenBank Accession Nos. NC_007622, NC_002951, NC_013450, and NC_009632 provide exemplary S. aureus genome sequences, all of which are incorporated by reference as provided by GenBank on Apr. 30, 2012.

The S. aureus gsf gene encodes a conserved region in glutamate synthase family protein. An exemplary S. aureus gsf nucleic acid sequence includes GenBank Accession No. CP003194 (2567647-2569224), which is incorporated by reference as provided by GenBank on Apr. 30, 2012. An exemplary Staphylococcus aureus nucleotide sequence of gsf is found at GenBank Accession No. CP003194 (2567647-2569224) (SEQ ID NO: 8). Streptococcus agalactiae (Group B Streptococcus; GBS): A gram-positive bacterium that is a major cause of meningitis and sepsis in neonates. GBS can also asymptomatically colonize skin and mucous membranes. Nucleic acid and protein sequences for GBS are publicly available. For example, GenBank Accession Nos. NC_007432, NC_004166, and NC_019048 provide exemplary GBS genome sequences, all of which are incorporated by reference as provided by GenBank on Feb. 15, 2013.

The GBS cfb gene encodes the CAMP factor. Exemplary GBS cfb nucleic acid sequences include GenBank Accession Nos. NC_004116 (2016473-2017240), NC_007432 (1969227-1969994), and NC_019048 (1695124-1695891), all of which are incorporated by reference herein as provided by GenBank on Feb. 15, 2013. An exemplary GBS nucleotide sequence of cfb is found at GenBank Accession No. NC_004116 (2016473-2017240) (SEQ ID NO: 53).

Toxoplasma gondii: A parasite of many animal species. Cats are the only host capable of passing infective T. gondii oocysts to subsequent hosts. Humans may become infected by exposure to contaminated undercooked meat or exposure to water, soil, or other material contaminated with T. gondii laden feline feces. Infection may be asymptomatic or elicit mild symptoms; however serious neurologic or ocular disease may result in the fetus of an exposed pregnant women. Nucleic acid and protein sequences for T. gondii are publicly available. For example, GenBank Accession No. NZ_ABPA00000000 provides an exemplary T. gondii genome sequence, which is incorporated by reference as provided by GenBank on Apr. 30, 2012.

The T. gondii ssrRNA gene is a small subunit ribosomal RNA. An exemplary T. gondii ssrRNA nucleic acid sequence includes GenBank Accession No. EF472967, which is incorporated herein by reference as present in GenBank on Apr. 30, 2012. An exemplary Toxoplasma gondii nucleotide sequence of ssrRNA is found at GenBank Accession No. EF472967 (SEQ ID NO: 9).

Ureaplasma parvum: Previously classified as Ureaplasma urealyticum biovar 1. A mycoplasma which can cause genito-urinary infection and infertility. It can also cause respiratory infection in neonates as a result of maternal transmission during delivery. Nucleic acid and protein sequences for U. parvum are publicly available. For example, GenBank Accession Nos. NC_010503 and NC_002162 provide exemplary U. parvum genome sequences, both of which are incorporated by reference as provided by GenBank on Apr. 30, 2012.

The U. parvum ure gene encodes the urease complex. An exemplary U. parvum ure nucleic acid sequence includes GenBank Accession No. AF085733, which is incorporated by reference as provided by GenBank on Apr. 30, 2012. An exemplary Ureaplasma parvum nucleotide sequence of ureC is found at GenBank Accession No. AF085733 (912-2708) (SEQ ID NO: 10).

Ureaplasma spp.: A genus of gram-negative bacteria which is urease positive. Ureaplasma spp. refers to any species in the genus Ureaplasma. In some embodiments, Ureaplasma spp. includes Ureaplasma parvum and Ureaplasma urealyticum. One of ordinary skill in the art can identify additional Ureaplasma spp. (such as U. canigenitalium, U. cati, U. diversum, U. felinum, U. gallorale, and U. loridis), for example utilizing the NCBI Taxonomy Browser (e.g., www.ncbi.nlm.nih.gov/Taxonomy/).

Ureaplasma urealyticum: A mycoplasma which can cause genito-urinary infection, infertility, and meningitis. It can also cause respiratory disease in neonates as a result of maternal transmission during delivery. Nucleic acid and protein sequences for U. urealyticum are publicly available. For example, GenBank Accession No. NC_011374 provides an exemplary U. urealyticum genome sequence, which is incorporated by reference as provided by GenBank on Apr. 30, 2012.

The U. urealyticum mba gene encodes the multiple banded antigen. An exemplary U. urealyticum mba nucleic acid sequence includes GenBank Accession No. AF055367, which is incorporated by reference as provided by GenBank on Apr. 30, 2012. An exemplary Ureaplasma urealyticum nucleotide sequence of mba is found at GenBank Accession No. AF055367 (SEQ ID NO: 11).

III. Methods for Detection of Pathogens

Methods for detecting the presence of a pathogen such as Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumooniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp. Streptococcus agalactiae, and/or Neisseria meningitidis in a sample are disclosed, for example, utilizing the probes and/or primers disclosed herein. In some embodiments, the methods include detection of a single selected pathogen. In other embodiments, the methods include detection of one or more of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcus agalactiae, Neisseria meningitidis or any combination of two or more thereof (for example, utilizing a simultaneous multipathogen detection assay (such as an array or card assay) or a multiplex assay).

The methods described herein may be used for any purpose for which detection of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcus agalactiae, and/or Neisseria meningitidis is desirable, including diagnostic or prognostic applications, such as in laboratory or clinical settings.

Appropriate samples include any conventional environmental or biological samples, including clinical samples obtained from a human or veterinary subject. Suitable samples include all biological samples useful for detection of infection in subjects, including, but not limited to, cells, tissues (for example, lung, liver, or kidney), autopsy samples, bone marrow aspirates, bodily fluids (for example, blood, serum, urine, cerebrospinal fluid, middle ear fluids, bronchoalveolar lavage, tracheal aspirates, sputum, nasopharyngeal swabs or aspirates, oropharyngeal swabs or aspirates, or saliva), eye swabs, cervical swabs, vaginal swabs, rectal swabs, stool, and stool suspensions. Suitable samples also include all samples useful for detection of a pathogen in an environment (such as a clinic or hospital), including but not limited to a water or fluid sample, a food sample, or a surface swab (for example, a swab of a counter, bed, floor, wall, or other surface). Standard techniques for acquisition of such samples are available. See for example, Schluger et al., J. Exp. Med. 176:1327-1333, 1992; Bigby et al., Am. Rev. Respir. Dis. 133:515-518, 1986; Kovacs et al., N. Engl. J. Med. 318:589-593, 1988; and Ognibene et al., Am. Rev. Respir. Dis. 129:929-932, 1984.

In some embodiments, the nucleic acids detected using the methods provided herein include nucleic acid molecules from Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumooniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcus agalactiae, or Neisseria meningitidis. In at least some embodiments, the disclosed methods can detect multiple strains or serotypes of a pathogen species. In some examples, the nucleic acids detected include nucleic acids from multidrug-resistant strains of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumooniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcus agalactiae, and/or Neisseria meningitidis. Strains of particular pathogens may be obtained from patient or environmental samples or laboratory or reference collections, for example, the American Type Culture Collection (Manassas, Va.). In one non-limiting example, the disclosed methods detect nucleic acids from methicillin-resistant S. aureus (MRSA), including hospital-acquired MRSA (HA-MRSA) or community-acquired MRSA (CA-MRSA). In other non-limiting examples, the disclosed methods detect nucleic acids from E. coli virotypes including enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), or enteroaggregative E. coli (EAEC).

One of ordinary skill in the art will know suitable methods for extracting nucleic acids such as RNA and/or DNA from a sample; such methods will depend upon, for example, the type of sample in which the pathogen nucleic acid is found. Nucleic acids can be extracted using standard methods. For instance, rapid nucleic acid preparation can be performed using a commercially available kit (such as kits and/or instruments from Qiagen (such as DNEASY® or RNEASY® kits), Roche Applied Science (such as MAGNA PURE® kits and instruments), Thermo Scientific (KingFisher mL), bioMérieux (NUCLISENS® NASBA Diagnostics), or Epicentre (MASTERPURE™ kits)). In other examples, the nucleic acids may be extracted using guanidinium isothiocyanate, such as single-step isolation by acid guanidinium isothiocyanate-phenol-chloroform extraction (Chomczynski et al. Anal. Biochem. 162:156-159, 1987). The sample can be used directly or can be processed, such as by adding solvents, preservatives, buffers, or other compounds or substances.

Detecting presence of at least one of an Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumooniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcus agalactiae, or Neisseria meningitidis nucleic acid in a sample involves contacting the sample with at least one of the probes (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 probes) disclosed herein that is capable of hybridizing to an Acinetobacter baumannii oxa-51 nucleic acid, a Pseudomonas aeruginosa gyrB nucleic acid, a Klebsiella pneumoniae nifA nucleic acid, a Toxoplasma gondii ssrRNA nucleic acid, a Moraxella catarrhalis purH nucleic acid, an Escherichia coli and/or Shigella uidA nucleic acid, a Staphylococcus aureus gsf nucleic acid, a Pneumocystis jirovecii dhps nucleic acid, a Chlamydia trachomatis tmRNA nucleic acid, a Ureaplasma urealyticum mba nucleic acid, a Ureaplasma parvum ure nucleic acid, a Ureaplasma spp. ure nucleic acid, a Bartonella spp. ssrA nucleic acid, a Streptococcus agalactiae cfb nucleic acid, or a Neisseria meningitidis sodC nucleic acid, for example, under conditions of high or very high stringency.

One of ordinary skill in the art can determine low, high, or very high stringency conditions for hybridization of a primer or probe (such as a probe or primer disclosed herein) to a nucleic acid sequence (for example to one of SEQ ID NOs: 1-11, 52, 53, 57, or 61). In some examples, the conditions are for hybridization of a primer or probe to a nucleic acid attached to a solid support (such as the conditions provided above). In other examples, the conditions are for hybridization of a primer or probe to a nucleic acid in solution, such as a PCR reaction mixture. In some non-limiting examples, low stringency conditions include hybridization (such as an annealing step in PCR) at a temperature of about 45-50° C. In other examples, high stringency conditions include hybridization (such as an annealing step in PCR) at a temperature of about 50-60° C. In further examples, very high stringency conditions include hybridization (such as an annealing step in PCR) at a temperature of greater than 60° C. One of skill in the art can determine appropriate hybridization or annealing conditions (including the degree of hybridization) based on the particular primers or probes and target nucleic acids to be amplified or detected.

In some embodiments, the methods include contacting the sample with at least one probe comprising a nucleic acid molecule between 10 and 40 nucleotides in length and detecting hybridization between the one or more probes and a nucleic acid in the sample, wherein detection of hybridization indicates the presence of one or more of said pathogens in the sample. In some examples, the probe is capable of hybridizing (such as under high stringency or very high stringency conditions) to an Acinetobacter baumannii nucleic acid sequence set forth as SEQ ID NO: 1, a Chlamydia trachomatis nucleic acid sequence set forth as SEQ ID NO: 2, an Escherichia coli nucleic acid sequence set forth as SEQ ID NO: 3, a Klebsiella pneumoniae nucleic acid sequence set forth as SEQ ID NO: 4 or SEQ ID NO: 57, a Moraxella catarrhalis nucleic acid sequence set forth as SEQ ID NO: 5, a Pneumocystis jirovecii nucleic acid sequence set forth as SEQ ID NO: 6, a Pseudomonas aeruginosa nucleic acid sequence set forth as SEQ ID NO: 7, a Staphylococcus aureus nucleic acid sequence set forth as SEQ ID NO: 8, a Toxoplasma gondii nucleic acid sequence set forth as SEQ ID NO: 9, a Ureaplasma parvum nucleic acid sequence set forth as SEQ ID NO: 10, a Ureaplasma urealyticum nucleic acid sequence set forth as SEQ ID NO: 11, a Bartonella spp. nucleic acid set forth as SEQ ID NO: 52, a Streptococcus agalactiae nucleic acid sequence set forth as SEQ ID NO: 53, a Neisseria meningitidis nucleic acid set forth as SEQ ID NO: 61, or a nucleic acid sequence at least 90% identical (for example 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical) to one of SEQ ID NOs: 1-11 52-53, 57, or 61. In some examples, the sample is contacted with one or more nucleic acid probes between 20 and 40 nucleotides in length comprising or consisting of a nucleic acid sequence set forth as any one of SEQ ID NOs: 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 56, 60, 64, or the reverse complement thereof.

In particular examples, the probes are detectably labeled (for example, as described in section IV, below). In some examples, the probes are at least 10, 15, 20, 25, 30, 35, or 40 nucleotides in length. In other examples, the probes may be no more than 10, 15, 20, 25, 30, 35, or 40 nucleotides in length. In further examples, the probes are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length.

Detection of hybridization between an Acinetobacter baumannii probe (for example SEQ ID NO: 14) and a nucleic acid indicates the presence of Acinetobacter baumannii nucleic acid in the sample, detection of hybridization between a Chlamydia trachomatis probe (for example SEQ ID NO: 38) and a nucleic acid indicates the presence of Chlamydia trachomatis nucleic acid in the sample, detection of hybridization between an Escherichia coli/Shigella probe (for example SEQ ID NO: 29) and a nucleic acid indicates the presence of E. coli and/or Shigella nucleic acid in the sample, detection of hybridization between a Klebsiella pneumoniae probe (for example SEQ ID NO: 20 or SEQ ID NO: 60) and a nucleic acid indicates the presence of Klebsiella pneumoniae nucleic acid in the sample, detection of hybridization between a Moraxella catarrhalis probe (for example SEQ ID NO: 26) and a nucleic acid indicates the presence of Moraxella catarrhalis nucleic acid in the sample, detection of hybridization between a Pneumocystis jirovecii probe (for example SEQ ID NO: 35) and a nucleic acid indicates the presence of Pneumocystis jirovecii nucleic acid in the sample, detection of hybridization between a Pseudomonas aeruginosa probe (for example SEQ ID NO: 17) and a nucleic acid indicates the presence of Pseudomonas aeruginosa nucleic acid in the sample, detection of hybridization between a Staphylococcus aureus probe (for example SEQ ID NO: 32) and a nucleic acid indicates the presence of Staphylococcus aureus nucleic acid in the sample, detection of hybridization between a Toxoplasma gondii probe (for example SEQ ID NO: 23) and a nucleic acid indicates the presence of Toxoplasma gondii nucleic acid in the sample, detection of hybridization between a Ureaplasma parvum probe (for example SEQ ID NO: 44) and a nucleic acid indicates the presence of Ureaplasma parvum nucleic acid in the sample, detection of hybridization between a Ureaplasma urealyticum probe (for example SEQ ID NO: 41) and a nucleic acid indicates the presence of Ureaplasma urealyticum nucleic acid in the sample, detection of hybridization between a Ureaplasma spp. probe (for example SEQ ID NO: 47) and a nucleic acid indicates the presence of Ureaplasma spp. nucleic acid in the sample, detection of hybridization between a Bartonella spp. probe (for example SEQ ID NO: 50) and a nucleic acid indicates the presence of Bartonella spp. nucleic acid in the sample, detection of hybridization between a Streptococcus agalactiae probe (for example SEQ ID NO: 56) and a nucleic acid indicates the presence of Streptococcus agalactiae nucleic acid in the sample, and detection of hybridization between a Neisseria meningitidis probe (for example SEQ ID NO: 64) and a nucleic acid indicates the presence of a Neisseria meningitidis nucleic acid in the sample.

In some embodiments, the methods disclosed herein further include positive and/or negative controls. One of ordinary skill in the art can select suitable controls. In some examples, a negative control is a no template control (such as a reaction that includes all components except the nucleic acid sample). In other examples, a positive control includes a sample known to include nucleic acid from a particular pathogen. In further examples, a positive control includes an internal positive control, such as a human nucleic acid (for example, RNase P) when the sample is from a human subject. In other examples, a positive control includes a synthetic positive control (such as a combined positive control), for example, a nucleic acid molecule including forward primer, probe, and reverse primer sequences for one or more primer/probe sets included in the assay. A combined positive control may also include additional positive or negative controls, such as a human nucleic acid control (for example, RNase P), and/or a control for laboratory contamination (such as a primer/probe that is not included in the assay). See, e.g., Kodani and Winchell (J. Clin. Microbiol. 50:1057-1060, 2011; incorporated herein by reference) for exemplary methods for constructing a combined positive control.

In some embodiments, nucleic acids present in a sample (for example, one or more of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcus agalactiae, or Neisseria meningitidis nucleic acids) are amplified prior to using a probe for detection. For instance, it can be advantageous to amplify a portion of one of more of the disclosed nucleic acids, and then detect the presence of the amplified nucleic acid, for example, to increase the number of nucleic acids that can be detected, thereby increasing the signal obtained. Specific nucleic acid primers can be used to amplify a region that is at least about 50, at least about 60, at least about 70, at least about 80 at least about 90, at least about 100, at least about 200, at least about 250, at least about 300, at least about 400, at least about 500, at least about 1000, at least about 2000, or more base pairs in length to produce amplified nucleic acids (such as amplified Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcus agalactiae, or Neisseria meningitidis nucleic acids). In other examples, specific nucleic acid primers can be used to amplify a region that is about 50-3000 base pairs in length (for example, about 70-2000 base pairs, about 100-1000 base pairs, about 50-300 base pairs, about 300-500 base pairs, or about 1000-3000 base pairs in length).

Detecting the amplified product typically includes the use of labeled probes that are sufficiently complementary to, and hybridize to, the amplified nucleic acid sequence. Thus, the presence, amount, and/or identity of the amplified product can be detected by hybridizing a labeled probe, such as a fluorescently labeled probe, complementary to the amplified product. In one embodiment, the detection of a target nucleic acid sequence of interest, such as an Acinetobacter baumannii oxa-51 nucleic acid, a Pseudomonas aeruginosa gyrB nucleic acid, a Klebsiella pneumoniae nifA nucleic acid, a Klebsiella pneumoniae diguanylate cyclase nucleic acid, a Toxoplasma gondii ssrRNA nucleic acid, a Moraxella catarrhalis purH nucleic acid, an Escherichia coli and/or Shigella uidA nucleic acid, a Staphylococcus aureus gsf nucleic acid, a Pneumocystis jirovecii dhps nucleic acid, a Chlamydia trachomatis tmRNA nucleic acid, a Ureaplasma urealyticum mba nucleic acid, a Ureaplasma parvum ure nucleic acid, a Ureaplasma spp. ure nucleic acid, a Bartonella spp. ssrA nucleic acid, a Streptococcus agalactiae cfb nucleic acid, or a Neisseria meningitidis sodC nucleic acid includes the combined use of PCR amplification and a labeled probe such that the product is measured using real-time PCR (such as TAQMAN® real-time PCR). In another embodiment, the detection of an amplified target nucleic acid sequence of interest includes the transfer of the amplified target nucleic acid to a solid support, such as a blot, for example a Northern blot, and probing the blot with a probe, for example a labeled probe, that is complementary to the amplified target nucleic acid. In still further embodiments, the detection of amplified target nucleic acid of interest includes the hybridization of a labeled amplified target nucleic acid to probes disclosed herein that are arrayed in a predetermined array with an addressable location and that are complementary to the amplified target nucleic acid.

Any nucleic acid amplification method can be used to detect the presence of one or more of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcus agalactiae, or Neisseria meningitidis nucleic acids in a sample. In one specific, non-limiting example, polymerase chain reaction (PCR) is used to amplify the pathogen-specific nucleic acid sequences. In other specific, non-limiting examples, real-time PCR, reverse transcriptase-polymerase chain reaction (RT-PCR), real-time reverse transcriptase-polymerase chain reaction (rt RT-PCR), ligase chain reaction, or transcription-mediated amplification (TMA) is used to amplify the nucleic acids. In a specific example, one or more (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) of an Acinetobacter baumannii oxa-51 nucleic acid, a Pseudomonas aeruginosa gyrB nucleic acid, a Klebsiella pneumoniae nifA nucleic acid, a Klebsiella pneumoniae diguanylate cyclase nucleic acid, a Toxoplasma gondii ssrRNA nucleic acid, a Moraxella catarrhalis purH nucleic acid, an Escherichia coli/Shigella uidA nucleic acid, a Staphylococcus aureus gsf nucleic acid, a Pneumocystis jirovecii dhps nucleic acid, a Chlamydia trachomatis tmRNA nucleic acid, a Ureaplasma urealyticum mba nucleic acid, a Ureaplasma parvum ure nucleic acid, a Ureaplasma spp. ure nucleic acid, a Bartonella spp. ssrA nucleic acid, a Streptococcus agalactiae cfb nucleic acid, a Neisseria meningitidis sodC nucleic acid, or any combination of two or more thereof are amplified by real-time PCR, for example real-time TAQMAN® PCR. Techniques for nucleic acid amplification are well-known to those of ordinary skill in the art.

Typically, at least two primers are utilized in the amplification reaction. In some examples, amplification of an Acinetobacter baumannii nucleic acid involves contacting the Acinetobacter baumannii nucleic acid with one or more primers (such as two or more primers) that are capable of hybridizing to and directing the amplification of an Acinetobacter baumannii nucleic acid, such as a primer capable of hybridizing under high or very high stringency conditions to an Acinetobacter baumannii nucleic acid sequence set forth as SEQ NO: 1, for example a primer that is least 90% identical (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleotide sequence set forth as one of SEQ ID NOs: 12 or 13. In one example, an Acinetobacter baumannii oxa-51 nucleic acid is amplified utilizing a pair of primers, such as a forward primer at least 90% identical to SEQ ID NO: 12 and a reverse primer at least 90% identical to SEQ ID NO: 13, such as a forward primer comprising or consisting essentially of SEQ ID NO: 12 and a reverse primer comprising or consisting essentially of SEQ ID NO: 13.

In other examples, amplification of a Chlamydia trachomatis nucleic acid involves contacting the Chlamydia trachomatis nucleic acid with one or more primers (such as two or more primers) that are capable of hybridizing to and directing the amplification of a Chlamydia trachomatis nucleic acid, such as a primer capable of hybridizing under high or very high stringency conditions to a Chlamydia trachomatis nucleic acid sequence set forth as SEQ NO: 2, for example a primer that is least 90% identical (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleotide sequence set forth as one of SEQ ID NOs: 36 or 37. In one example, a Chlamydia trachomatis tmRNA nucleic acid is amplified utilizing a pair of primers, such as a forward primer at least 90% identical to SEQ ID NO: 36 and a reverse primer at least 90% identical to SEQ ID NO: 37, such as a forward primer comprising or consisting essentially of SEQ ID NO: 36 and a reverse primer comprising or consisting essentially of SEQ ID NO: 37.

In further examples, amplification of an Escherichia coli and/or Shigella nucleic acid involves contacting the Escherichia coli and/or Shigella nucleic acid with one or more primers (such as two or more primers) that are capable of hybridizing to and directing the amplification of an Escherichia coli and/or Shigella nucleic acid, such as a primer capable of hybridizing under high or very high stringency conditions to an Escherichia coli and/or Shigella nucleic acid sequence set forth as SEQ NO: 3, for example a primer that is least 90% identical (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleotide sequence set forth as one of SEQ ID NOs: 27 or 28. In one example, an Escherichia coli uidA nucleic acid is amplified utilizing a pair of primers, such as a forward primer at least 90% identical to SEQ ID NO: 27 and a reverse primer at least 90% identical to SEQ ID NO: 28, such as a forward primer comprising or consisting essentially of SEQ ID NO: 27 and a reverse primer comprising or consisting essentially of SEQ ID NO: 28.

In some examples, amplification of a Klebsiella pneumoniae nucleic acid involves contacting the Klebsiella pneumoniae nucleic acid with one or more primers (such as two or more primers) that are capable of hybridizing to and directing the amplification of a Klebsiella pneumoniae nucleic acid, such as a primer capable of hybridizing under high or very high stringency conditions to a Klebsiella pneumoniae nucleic acid sequence set forth as SEQ NO: 4, for example a primer that is least 90% identical (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleotide sequence set forth as one of SEQ ID NOs: 18 or 19. In one example, a Klebsiella pneumoniae nifA nucleic acid is amplified utilizing a pair of primers, such as a forward primer at least 90% identical to SEQ ID NO: 18 and a reverse primer at least 90% identical to SEQ ID NO: 19, such as a forward primer comprising or consisting essentially of SEQ ID NO: 18 and a reverse primer comprising or consisting essentially of SEQ ID NO: 19. In other examples, amplification of a Klebsiella pneumoniae nucleic acid involves contacting the Klebsiella pneumoniae nucleic acid with one or more primers (such as two or more primers) that are capable of hybridizing to and directing the amplification of a Klebsiella pneumoniae nucleic acid, such as a primer capable of hybridizing under high or very high stringency conditions to a Klebsiella pneumoniae nucleic acid sequence set forth as SEQ NO: 57, for example a primer that is least 90% identical (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleotide sequence set forth as one of SEQ ID NOs: 58 or 59. In one example, a Klebsiella pneumoniae diguanylate cyclase nucleic acid is amplified utilizing a pair of primers, such as a forward primer at least 90% identical to SEQ ID NO: 58 and a reverse primer at least 90% identical to SEQ ID NO: 59, such as a forward primer comprising or consisting essentially of SEQ ID NO: 58 and a reverse primer comprising or consisting essentially of SEQ ID NO: 59.

In additional examples, amplification of a Moraxella catarrhalis nucleic acid involves contacting the Moraxella catarrhalis nucleic acid with one or more primers (such as two or more primers) that are capable of hybridizing to and directing the amplification of a Moraxella catarrhalis nucleic acid, such as a primer capable of hybridizing under high or very high stringency conditions to a Moraxella catarrhalis nucleic acid sequence set forth as SEQ NO: 5, for example a primer that is least 90% identical (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleotide sequence set forth as one of SEQ ID NOs: 24 or 25. In one example, a Moraxella catarrhalis purH nucleic acid is amplified utilizing a pair of primers, such as a forward primer at least 90% identical to SEQ ID NO: 24 and a reverse primer at least 90% identical to SEQ ID NO: 25, such as a forward primer comprising or consisting essentially of SEQ ID NO: 24 and a reverse primer comprising or consisting essentially of SEQ ID NO: 25.

In additional examples, amplification of a Pneumocystis jirovecii nucleic acid involves contacting the Pneumocystis jirovecii nucleic acid with one or more primers (such as two or more primers) that are capable of hybridizing to and directing the amplification of a Pneumocystis jirovecii nucleic acid, such as a primer capable of hybridizing under high or very high stringency conditions to a Pneumocystis jirovecii nucleic acid sequence set forth as SEQ NO: 6, for example a primer that is least 90% identical (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleotide sequence set forth as one of SEQ ID NOs: 33 or 34. In one example, a P. jirovecii dhps nucleic acid is amplified utilizing a pair of primers, such as a forward primer at least 90% identical to SEQ ID NO: 33 and a reverse primer at least 90% identical to SEQ ID NO: 34, such as a forward primer comprising or consisting essentially of SEQ ID NO: 33 and a reverse primer comprising or consisting essentially of SEQ ID NO: 34.

In further examples, amplification of a Pseudomonas aeruginosa nucleic acid involves contacting the Pseudomonas aeruginosa nucleic acid with one or more primers (such as two or more primers) that are capable of hybridizing to and directing the amplification of a Pseudomonas aeruginosa nucleic acid, such as a primer capable of hybridizing under high or very high stringency conditions to a Pseudomonas aeruginosa nucleic acid sequence set forth as SEQ NO: 7, for example a primer that is least 90% identical (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleotide sequence set forth as one of SEQ ID NOs: 15 or 16. In one example, a Pseudomonas aeruginosa gyrB nucleic acid is amplified utilizing a pair of primers, such as a forward primer at least 90% identical to SEQ ID NO: 15 and a reverse primer at least 90% identical to SEQ ID NO: 16, such as a forward primer comprising or consisting essentially of SEQ ID NO: 15 and a reverse primer comprising or consisting essentially of SEQ ID NO: 16.

In still further examples, amplification of a Staphylococcus aureus nucleic acid involves contacting the Staphylococcus aureus nucleic acid with one or more primers (such as two or more primers) that are capable of hybridizing to and directing the amplification of a Staphylococcus aureus nucleic acid, such as a primer capable of hybridizing under high or very high stringency conditions to a Staphylococcus aureus nucleic acid sequence set forth as SEQ NO: 8, for example a primer that is least 90% identical (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleotide sequence set forth as one of SEQ ID NOs: 30 or 31. In one example, a Staphylococcus aureus gsf nucleic acid is amplified utilizing a pair of primers, such as a forward primer at least 90% identical to SEQ ID NO: 30 and a reverse primer at least 90% identical to SEQ ID NO: 31, such as a forward primer comprising or consisting essentially of SEQ ID NO: 30 and a reverse primer comprising or consisting essentially of SEQ ID NO: 31.

In other examples, amplification of a Toxoplasma gondii nucleic acid involves contacting the Toxoplasma gondii nucleic acid with one or more primers (such as two or more primers) that are capable of hybridizing to and directing the amplification of a Toxoplasma gondii nucleic acid, such as a primer capable of hybridizing under high or very high stringency conditions to a Toxoplasma gondii nucleic acid sequence set forth as SEQ NO: 9, for example a primer that is least 90% identical (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleotide sequence set forth as one of SEQ ID NOs: 21 or 22. In one example, a Toxoplasma gondii ssrRNA nucleic acid is amplified utilizing a pair of primers, such as a forward primer at least 90% identical to SEQ ID NO: 21 and a reverse primer at least 90% identical to SEQ ID NO: 22, such as a forward primer comprising or consisting essentially of SEQ ID NO: 21 and a reverse primer comprising or consisting essentially of SEQ ID NO: 22.

In additional examples, amplification of a Ureaplasma parvum nucleic acid involves contacting the Ureaplasma parvum nucleic acid with one or more primers (such as two or more primers) that are capable of hybridizing to and directing the amplification of a Ureaplasma parvum nucleic acid, such as a primer capable of hybridizing under high or very high stringency conditions to a Ureaplasma parvum nucleic acid sequence set forth as SEQ NO: 10, for example a primer that is least 90% identical (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleotide sequence set forth as one of SEQ ID NOs: 42 or 43. In one example, a Ureaplasma parvum ure nucleic acid is amplified utilizing a pair of primers, such as a forward primer at least 90% identical to SEQ ID NO: 42 and a reverse primer at least 90% identical to SEQ ID NO: 43, such as a forward primer comprising or consisting essentially of SEQ ID NO: 42 and a reverse primer comprising or consisting essentially of SEQ ID NO: 43.

In further examples, amplification of a Ureaplasma urealyticum nucleic acid involves contacting the Ureaplasma urealyticum nucleic acid with one or more primers (such as two or more primers) that are capable of hybridizing to and directing the amplification of a Ureaplasma urealyticum nucleic acid, such as a primer capable of hybridizing under high or very high stringency conditions to a Ureaplasma urealyticum nucleic acid sequence set forth as SEQ NO: 11, for example a primer that is least 90% identical (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleotide sequence set forth as one of SEQ ID NOs: 39 or 40. In one example, a Ureaplasma urealyticum mba nucleic acid is amplified utilizing a pair of primers, such as a forward primer at least 90% identical to SEQ ID NO: 39 and a reverse primer at least 90% identical to SEQ ID NO: 40, such as a forward primer comprising or consisting essentially of SEQ ID NO: 39 and a reverse primer comprising or consisting essentially of SEQ ID NO: 40.

In additional examples, amplification of a Ureaplasma spp. nucleic acid involves contacting the Ureaplasma spp. nucleic acid with one or more primers (such as two or more primers) that are capable of hybridizing to and directing the amplification of a Ureaplasma spp. nucleic acid, such as a primer capable of hybridizing under very high stringency conditions to a Ureaplasma spp. nucleic acid sequence set forth as SEQ NO: 10, for example a primer that is least 90% identical (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleotide sequence set forth as one of SEQ ID NOs: 45 or 46. In one example, a Ureaplasma spp. ure nucleic acid is amplified utilizing a pair of primers, such as a forward primer at least 90% identical to SEQ ID NO: 45 and a reverse primer at least 90% identical to SEQ ID NO: 46, such as a forward primer comprising or consisting essentially of SEQ ID NO: 45 and a reverse primer comprising or consisting essentially of SEQ ID NO: 46.

In further examples, amplification of a Bartonella spp. nucleic acid involves contacting the Bartonella spp. nucleic acid with one or more primers (such as two or more primers) that are capable of hybridizing to and directing the amplification of a Bartonella spp. nucleic acid, such as a primer capable of hybridizing under high or very high stringency conditions to a Bartonella spp. ssrA nucleic acid sequence (such as SEQ ID NO: 52), for example a primer that is least 90% identical (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleotide sequence set forth as one of SEQ ID NOs: 48, 49, or 51. In one example, a Bartonella spp. ssrA nucleic acid is amplified utilizing a pair of primers, such as a forward primer at least 90% identical to SEQ ID NO: 48 and a reverse primer at least 90% identical to SEQ ID NO: 49, such as a forward primer comprising or consisting essentially of SEQ ID NO: 48 and a reverse primer comprising or consisting essentially of SEQ ID NO: 49. In other examples, a Bartonella spp. ssrA nucleic acid is amplified utilizing a pair of primers, such as a forward primer at least 90% identical to SEQ ID NO: 51 and a reverse primer at least 90% identical to SEQ ID NO: 49, such as a forward primer comprising or consisting essentially of SEQ ID NO: 51 and a reverse primer comprising or consisting essentially of SEQ ID NO: 49.

In other examples, amplification of a Streptococcus agalactiae nucleic acid involves contacting the Streptococcus agalactiae nucleic acid with one or more primers (such as two or more primers) that are capable of hybridizing to and directing the amplification of a Streptococcus agalactiae nucleic acid, such as a primer capable of hybridizing under high or very high stringency conditions to a Streptococcus agalactiae nucleic acid sequence set forth as SEQ NO: 53, for example a primer that is least 90% identical (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleotide sequence set forth as one of SEQ ID NOs: 54 or 55. In one example, a Streptococcus agalactiae cfb nucleic acid is amplified utilizing a pair of primers, such as a forward primer at least 90% identical to SEQ ID NO: 54 and a reverse primer at least 90% identical to SEQ ID NO: 55, such as a forward primer comprising or consisting essentially of SEQ ID NO: 54 and a reverse primer comprising or consisting essentially of SEQ ID NO: 55.

In other examples, amplification of a Neisseria meningitidis nucleic acid involves contacting the Neisseria meningitidis nucleic acid with one or more primers (such as two or more primers) that are capable of hybridizing to and directing the amplification of a Neisseria meningitidis nucleic acid, such as a primer capable of hybridizing under high or very high stringency conditions to a Neisseria meningitidis nucleic acid sequence set forth as SEQ NO: 61, for example a primer that is least 90% identical (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleotide sequence set forth as one of SEQ ID NOs: 62 or 63. In one example, a Neisseria meningitidis sodC nucleic acid is amplified utilizing a pair of primers, such as a forward primer at least 90% identical to SEQ ID NO: 62 and a reverse primer at least 90% identical to SEQ ID NO: 63, such as a forward primer comprising or consisting essentially of SEQ ID NO: 62 and a reverse primer comprising or consisting essentially of SEQ ID NO: 63.

The amplified Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumooniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli/Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum ure, Ureaplasma spp. Bartonella spp., Streptococcus agalactiae, or Neisseria meningitidis nucleic acid can be detected in real-time, for example by real-time PCR, in order to determine the presence and/or the amount of a pathogen nucleic acid in a sample. In this manner, an amplified nucleic acid sequence can be detected using a probe specific for the product amplified from the target sequence of interest. Suitable probes for real-time PCR include those described herein, such as a probe having a nucleic acid sequence at least 90% identical to SEQ ID NO: 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 56, 60, or 64. In particular examples of the disclosed methods, multiplex real-time PCR is utilized to detect one or more (for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) of an Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumooniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli/Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp. Bartonella spp., Streptococcus agalactiae, or Neisseria meningitidis nucleic acid present in the sample. In other examples of the disclosed methods, simultaneous multipathogen detection (such as multiple singleplex real-time PCR reactions, for example on a single array or card) is utilized to detect one or more (for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) of an Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumooniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli/Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp. Bartonella spp., Streptococcus agalactiae, or Neisseria meningitidis nucleic acid present in the sample.

Real-time PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon production during each PCR cycle, as opposed to endpoint detection. The real-time progress of the reaction can be viewed in some systems. Typically, real-time PCR uses the detection of a fluorescent reporter. Typically, the fluorescent reporter's signal increases in direct proportion to the amount of PCR product in a reaction. By recording the amount of fluorescence emission at each cycle, it is possible to monitor the PCR reaction during exponential phase where the first significant increase in the amount of PCR product correlates to the initial amount of target template. The higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed.

In one embodiment, the fluorescently-labeled probes (such as probes disclosed herein) rely upon fluorescence resonance energy transfer (FRET), or in a change in the fluorescence emission wavelength of a sample, as a method to detect hybridization of a DNA probe to the amplified target nucleic acid in real-time. For example, FRET that occurs between fluorogenic labels on different probes (for example, using HybProbes) or between a donor fluorophore and an acceptor or quencher fluorophore on the same probe (for example, using a molecular beacon or a TAQMAN® probe) can identify a probe that specifically hybridizes to the nucleic acid of interest and in this way, using an Acinetobacter baumannii oxa-51 probe, a Pseudomonas aeruginosa gyrB probe, a Klebsiella pneumoniae nifA probe or a Klebsiella pneumoniae diguanylate cyclase probe, a Toxoplasma gondii ssrRNA probe, a Moraxella catarrhalis purH probe, an Escherichia coli/Shigella uidA probe, a Staphylococcus aureus gsf probe, a Pneumocystis jirovecii dhps probe, a Chlamydia trachomatis tmRNA probe, a Ureaplasma urealyticum mba probe, a Ureaplasma parvum ure probe, a Ureaplasma spp. ure probe, a Bartonella spp. ssrA probe, a Streptococcus agalactiae cfb probe, or a Neisseria meningitidis sodC probe can detect the presence and/or amount of the respective pathogen in a sample.

In some embodiments, the fluorescently-labeled DNA probes used to identify amplification products have spectrally distinct emission wavelengths, thus allowing them to be distinguished within the same reaction tube, for example in multiplex PCR, such as a multiplex real-time PCR. In some embodiments, the probes and primers disclosed herein are used in multiplex real-time PCR. For example, multiplex PCR permits the simultaneous detection of one or more (for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) of the amplification products of Acinetobacter baumannii oxa-51 nucleic acid, a Pseudomonas aeruginosa gyrB nucleic acid, a Klebsiella pneumoniae nifA nucleic acid, a Klebsiella pneumoniae diguanylate cyclase nucleic acid, a Toxoplasma gondii ssrRNA nucleic acid, a Moraxella catarrhalis purH nucleic acid, an Escherichia coli/Shigella uidA nucleic acid, a Staphylococcus aureus gsf nucleic acid, a Pneumocystis jirovecii dhps nucleic acid, a Chlamydia trachomatis tmRNA nucleic acid, a Ureaplasma urealyticum mba nucleic acid, a Ureaplasma parvum ure nucleic acid, a Ureaplasma spp. ure nucleic acid, a Bartonella spp. ssrA nucleic acid, a Streptococcus agalactiae cfb nucleic acid, and a Neisseria meningitidis sodC nucleic acid. Using the disclosed primers and probes, any combination of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli/Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum ure, Ureaplasma spp., Bartonella spp. Streptococcus agalactiae, and Neisseria meningitidis nucleic acids can be detected. In some examples, the multiplex reaction may include one or more of the primers and probes disclosed herein and primers and probes for detection of additional pathogens.

In other examples, the probes and primers disclosed herein are used in a bead-based multiplex assay (see, e.g., U.S. Pat. No. 6,939,720). For example, probes specific for each pathogen (such as the probes disclosed herein) are attached to different fluorescently labeled beads and are hybridized to amplified DNA from the sample. The probes will only significantly hybridize if the particular pathogen is present in the sample. The hybridized beads are then captured, for example with a biotinylated detector molecule, and the relative fluorescence of the beads for each label is measured.

In another embodiment, a melting curve analysis of the amplified target nucleic acid can be performed subsequent to the amplification process. The T_(m) of a nucleic acid sequence depends on the length of the sequence and its G/C content. Thus, the identification of the T_(m) for a nucleic acid sequence can be used to identify the amplified nucleic acid, for example by using double-stranded DNA binding dye chemistry, which quantitates the amplicon production by the use of a non-sequence specific fluorescent intercalating agent (such as SYBR® Green or ethidium bromide). SYBR® Green is a fluorogenic minor groove binding dye that exhibits little fluorescence when in solution but emits a strong fluorescent signal upon binding to double-stranded DNA. Typically, SYBR® Green is used in singleplex reactions, however when coupled with melting point analysis, it can be used for multiplex reactions.

Any type of thermal cycler apparatus can be used for the amplification of pathogen or control nucleic acids and/or the determination of hybridization. Examples of suitable apparatuses include PTC-100® Peltier Thermal Cycler (MJ Research, Inc.; San Francisco, Calif.), ROBOCYCLER® 40 Temperature Cycler (Agilent/Stratagene; Santa Clara, Calif.), or GENEAMP® PCR System 9700 (Applied Biosystems; Foster City, Calif.). For real-time PCR, any type of real-time thermocycler apparatus can be used. For example, iCYCLER iQ™ or CFX96™ real-time detection systems (Bio-Rad, Hercules, Calif.), LightCycler® systems (Roche, Mannheim, Germany), ABI™ systems such as the 7000, 7300, 7500, 7700, or 7900 systems or the VIIA™ 7 real-time PCR system (Applied Biosystems; Foster City, Calif.), MX4000™, MX3000™ or MX3005™ qPCR systems (Agilent/Stratagene; Santa Clara, Calif.), DNA Engine OPTICON® Continuous Fluorescence Detection System (Bio-Rad, Hercules, Calif.), ROTOR-GENE® Q real-time cycler (Qiagen, Valencia, Calif.), or SMARTCYCLER® system (Cepheid, Sunnyvale, Calif.) can be used to amplify nucleic acid sequences in real-time. In some embodiments, real-time PCR is performed using a TAQMAN® array format, for example, a microfluidic card in which each well is pre-loaded with primers and probes for a particular target. The reaction is initiated by adding a sample including nucleic acids and assay reagents (such as a PCR master mix) and running the reactions in a real-time thermocycler apparatus.

In some embodiments, a microfluidic card includes at least one well containing primers and probes for at least one of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumooniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli/Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum ure, Ureaplasma spp. ure, Bartonella spp. ssrA nucleic acid, Streptococcus agalactiae, Neisseria meningitidis, or any combination or two or more thereof (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 thereof). In one non-limiting example, the card includes at least one well containing Acinetobacter baumannii primers and probe (e.g., SEQ ID NOs: 12-14), at least one well containing Pseudomonas aeruginosa primers and probe (e.g., SEQ ID NOs: 15-17), at least one well containing Klebsiella pneumoniae primers and probe (e.g., SEQ ID NOs: 18-20 or SEQ ID NOs: 58-60), at least one well containing Toxoplasma gondii primers and probe (e.g., SEQ ID NOs: 21-23), at least one well containing Moraxella catarrhalis primers and probe (e.g., SEQ ID NOs: 24-26), at least one well containing E. coli/Shigella primers and probe (e.g., SEQ ID NOs: 27-29), at least one well containing Staphylococcus aureus primers and probe (e.g., SEQ ID NOs: 30-32), at least one well containing Pneumocystis jirovecii primers and probe (e.g., SEQ ID NOs: 33-35), at least one well containing Chlamydia trachomatis primers and probe (e.g., SEQ ID NOs: 36-38), at least one well containing Ureaplasma urealyticum primers and probe (e.g., SEQ ID NOs: 39-41), at least one well containing Ureaplasma parvum primers and probe (e.g., SEQ ID NOs: 42-44), at least one well containing Ureaplasma spp. primers and probe (e.g., SEQ ID NOs: 45-47), at least one well containing Bartonella spp. primers and probe (e.g., SEQ ID NOs: 48-50 and/or SEQ ID NOs: 49-51), at least one well containing Streptococcus agalactiae primers and probe (e.g., SEQ ID NOs: 54-56), and at least one well containing Neisseria meningitidis primers and probe (e.g., SEQ ID NOs: 62-64).

In another non-limiting example, the card includes at least one well containing Chlamydia trachomatis primers and probe (e.g., SEQ ID NOs: 36-38) and at least one well containing Ureaplasma spp. primers and probe (e.g., SEQ ID NOs: 45-47). In yet another non-limiting example, the card includes at least one well containing Staphylococcus aureus primers and probe (e.g., SEQ ID NOs: 30-32), at least one well containing Pseudomonas aeruginosa primers and probe (e.g., SEQ ID NOs: 15-17), at least one well containing E. coli/Shigella primers and probe (e.g., SEQ ID NOs: 27-29), at least one well containing Klebsiella pneumoniae primers and probe (e.g., SEQ ID NOs: 18-20), at least one well containing Acinetobacter baumannii primers and probe (e.g., SEQ ID NOs: 12-14), at least one well containing Toxoplasma gondii primers and probe (e.g., SEQ ID NOs: 21-23), at least one well containing Ureaplasma spp. primers and probe (e.g., SEQ ID NOs: 45-47), at least one well containing Chlamydia trachomatis primers and probe (e.g., SEQ ID NOs: 36-38), and at least one well containing Streptococcus agalactiae primers and probe (e.g., SEQ ID NOs: 54-56). In some examples, this card may also include at least one well containing Neisseria meningitidis primers and probe (e.g., SEQ ID NOs: 62-64) and/or at least one well containing Salmonella spp. primers and probe (e.g., SEQ ID NOs: 65-67). The card may include additional primers and probes in additional wells, such as positive control primers and probes, or primers and probes for additional pathogens or other nucleic acids of interest. In some examples, a card may additionally include wells containing primers and probes for one or more of Mycoplasma pneumoniae, Chlamydophila pneumoniae, Bordetella pertussis, adenovirus, influenza virus (A or B), parainfluenza virus (type 1, 2, or 3), respiratory syncytial virus, parechovirus, enterovirus, human metapneumovirus, rubella, Streptococcus pneumoniae, Streptococcus pyogenes, rhinovirus, Group B Streptococcus, Herpes simplex virus (1 or 2), pan-Haemophilus influenzae, pan-Salmonella, Neisseria meningitidis, cytomegalovirus, or any combination of two or more thereof. Additional combinations of assays can be selected and included on a TAC, as will be understood by one of ordinary skill in the art.

In some embodiments, the probe is detectably labeled, either with an isotopic or non-isotopic label; in alternative embodiments, the target nucleic acid is labeled. Non-isotopic labels can, for instance, comprise a fluorescent or luminescent molecule, or an enzyme, co-factor, enzyme substrate, or hapten. The probe is incubated with a single-stranded or double-stranded preparation of RNA, DNA, or a mixture of both, and hybridization is determined. In some examples, the hybridization results in a detectable change in signal such as in increase or decrease in signal, for example from the labeled probe. Thus, detecting hybridization comprises detecting a change in signal from the labeled probe during or after hybridization relative to signal from the label before hybridization.

In some examples, the disclosed methods can predict with a sensitivity of at least 90% and a specificity of at least 90% for presence of an Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumooniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcus agalactiae, and/or Neisseria meningitidis nucleic acid, such as a sensitivity of at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% and a specificity of at least of at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100%.

IV. Probes and Primers

Probes and primers suitable for use in the disclosed methods are described herein. Such probes and primers include nucleic acid molecules capable of hybridizing to the disclosed nucleic acid molecules, such as SEQ ID NOs: 1-11 or 52.

A. Probes

Probes capable of hybridizing to and detecting the presence of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumooniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcus agalactiae, or Neisseria meningitidis nucleic acid molecules are disclosed. In some embodiments, the disclosed probes are between 10 and 40 nucleotides in length, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 29, 30, 31, 32, 32, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length and are capable of hybridizing to the disclosed nucleic acid molecules. In some examples, the probes are at least 10, 15, 20, 25, 30, 35, or 40 nucleotides in length. In other examples, the probes may be no more than 10, 15, 20, 25, 30, 35, or 40 nucleotides in length. The disclosed probes may also include a 3′ C6 CpG in some examples.

In several embodiments, a probe is capable of hybridizing under high or very high stringency conditions to an Acinetobacter baumannii nucleic acid sequence set forth as SEQ ID NO: 1. In some examples, a probe capable of hybridizing to an Acinetobacter baumannii oxa-51 nucleic molecule includes a nucleic acid sequence that is at least 90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleotide sequence set forth as TGACTGCTAATCCAAATCACAGCGCTTCA (SEQ ID NO: 14). In several embodiments, a probe capable of hybridizing to an Acinetobacter baumannii oxa-51 nucleic acid molecule consists essentially of, or consists of, a nucleic acid sequence set forth as SEQ ID NO: 14.

In some embodiments, a probe is capable of hybridizing under high or very high stringency conditions to a Chlamydia trachomatis nucleic acid sequence set forth as SEQ ID NO: 2. In some examples, a probe capable of hybridizing to a Chlamydia trachomatis tmRNA nucleic molecule includes a nucleic acid sequence that is at least 90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleotide sequence set forth as ATGCGGAGGGCGTTGGCTGG (SEQ ID NO: 38). In several embodiments, a probe capable of hybridizing to a Chlamydia trachomatis tmRNA nucleic acid molecule consists essentially of, or consists of, a nucleic acid sequence set forth as SEQ ID NO: 38.

In other embodiments, a probe is capable of hybridizing under high or very high stringency conditions to an Escherichia coli and/or Shigella spp. nucleic acid sequence set forth as SEQ ID NO: 3. In some examples, a probe capable of hybridizing to an Escherichia coli and/or Shigella spp. nucleic molecule includes a nucleic acid sequence that is at least 90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleotide sequence set forth as TACGGCGTGACATCGGCTTCAAATG (SEQ ID NO: 29). In several embodiments, a probe capable of hybridizing to an Escherichia coli uidA nucleic acid molecule consists essentially of, or consists of, a nucleic acid sequence set forth as SEQ ID NO: 29. In particular embodiments, the probe capable of hybridizing to an Escherichia coli uidA nucleic acid molecule is capable of hybridizing to a uidA nucleic acid molecule from any an Escherichia coli and/or Shigella species or serogroup.

In additional embodiments, a probe is capable of hybridizing under high or very high stringency conditions to a Klebsiella pneumoniae nucleic acid sequence set forth as SEQ ID NO: 4. In some examples, a probe capable of hybridizing to a Klebsiella pneumoniae nifA nucleic molecule includes a nucleic acid sequence that is at least 90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleotide sequence set forth as ACGCTGAGCACCTCCTGCAACGT (SEQ ID NO: 20). In several embodiments, a probe capable of hybridizing to a Klebsiella pneumoniae nifA nucleic acid molecule consists essentially of, or consists of, a nucleic acid sequence set forth as SEQ ID NO: 20. In other embodiments, a probe is capable of hybridizing under high or very high stringency conditions to a Klebsiella pneumoniae nucleic acid sequence set forth as SEQ ID NO: 57. In some examples, a probe capable of hybridizing to a Klebsiella pneumoniae diguanylate cyclase nucleic molecule includes a nucleic acid sequence that is at least 90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleotide sequence set forth as CCACCACGCTCATCTGTTTCGCC (SEQ ID NO: 60). In several embodiments, a probe capable of hybridizing to a Klebsiella pneumoniae diguanylate cyclase nucleic acid molecule consists essentially of, or consists of, a nucleic acid sequence set forth as SEQ ID NO: 60.

In further embodiments, a probe is capable of hybridizing under high or very high stringency conditions to a Moraxella catarrhalis nucleic acid sequence set forth as SEQ ID NO: 5. In some examples, a probe capable of hybridizing to a Moraxella catarrhalis purH nucleic molecule includes a nucleic acid sequence that is at least 90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleotide sequence set forth as CACAGCGGGCAGCTCAATTTGACCTA (SEQ ID NO: 26). In several embodiments, a probe capable of hybridizing to a Moraxella catarrhalis purH nucleic acid molecule consists essentially of, or consists of, a nucleic acid sequence set forth as SEQ ID NO: 26.

In still further embodiments, a probe is capable of hybridizing under high or very high stringency conditions to a Pneumocystis jirovecii nucleic acid sequence set forth as SEQ ID NO: 6. In some examples, a probe capable of hybridizing to a Pneumocystis jirovecii dhps nucleic molecule includes a nucleic acid sequence that is at least 90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleotide sequence set forth as ACAGGGTGTCTTACAGGTGATGTTATGCCAAAAG (SEQ ID NO: 35). In several embodiments, a probe capable of hybridizing to a Pneumocystis jirovecii dhps nucleic acid molecule consists essentially of, or consists of, a nucleic acid sequence set forth as SEQ ID NO: 35. In one example, a probe capable of hybridizing to a Pneumocystis jirovecii dhps nucleic acid molecule includes or consists of a nucleic acid sequence set forth as ACAGGGTGTCT“T”ACAGGTGATGTTATGCCAAAAG (SEQ ID NO: 35), where “T” is BHQ1.

In additional embodiments, a probe is capable of hybridizing under high or very high stringency conditions to a Pseudomonas aeruginosa nucleic acid sequence set forth as SEQ ID NO: 7. In some examples, a probe capable of hybridizing to a Pseudomonas aeruginosa gyrB nucleic molecule includes a nucleic acid sequence that is at least 90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleotide sequence set forth as TCCGTCGCCACAACAAGGTCTGGGAA (SEQ ID NO: 17). In several embodiments, a probe capable of hybridizing to a Pseudomonas aeruginosa gyrB nucleic acid molecule consists essentially of, or consists of, a nucleic acid sequence set forth as SEQ ID NO: 17.

In other embodiments, a probe is capable of hybridizing under high or very high stringency conditions to a Staphylococcus aureus nucleic acid sequence set forth as SEQ ID NO: 8. In some examples, a probe capable of hybridizing to a Staphylococcus aureus gsf nucleic molecule includes a nucleic acid sequence that is at least 90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleotide sequence set forth as TTCCATATGACCACCACGAGTCTTAGCACC (SEQ ID NO: 32). In several embodiments, a probe capable of hybridizing to a Staphylococcus aureus gsf nucleic acid molecule consists essentially of, or consists of, a nucleic acid sequence set forth as SEQ ID NO: 32.

In some embodiments, a probe is capable of hybridizing under high or very high stringency conditions to a Toxoplasma gondii nucleic acid sequence set forth as SEQ ID NO: 9. In some examples, a probe capable of hybridizing to a Toxoplasma gondii ssrRNA nucleic molecule includes a nucleic acid sequence that is at least 90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleotide sequence set forth as ATCGCGTTGACTTCGGTCTGCGAC (SEQ ID NO: 23). In several embodiments, a probe capable of hybridizing to a Toxoplasma gondii ssrRNA nucleic acid molecule consists essentially of, or consists of, a nucleic acid sequence set forth as SEQ ID NO: 23.

In some embodiments, a probe is capable of hybridizing under high or very high stringency conditions to a Ureaplasma parvum nucleic acid sequence set forth as SEQ ID NO: 10. In some examples, a probe capable of hybridizing to a Ureaplasma parvum ure nucleic molecule includes a nucleic acid sequence that is at least 90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleotide sequence set forth as TCAGTGAACGTGAGTATCTAAACCACCAGC (SEQ ID NO: 44). In several embodiments, a probe capable of hybridizing to a Ureaplasma parvum ure nucleic acid molecule consists essentially of, or consists of, a nucleic acid sequence set forth as SEQ ID NO: 44. In some examples, a probe capable of hybridizing to a Ureaplasma parvum ure nucleic acid molecule includes or consists of a nucleic acid sequence set forth as TCAGTGAACG“T”GAGTATCTAAACCACCAGC (SEQ ID NO: 44), where “T” is BHQ1.

In additional embodiments, a probe is capable of hybridizing under high or very high stringency conditions to a Ureaplasma urealyticum nucleic acid sequence set forth as SEQ ID NO: 11. In some examples, a probe capable of hybridizing to a Ureaplasma urealyticum mba nucleic molecule includes a nucleic acid sequence that is at least 90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleotide sequence set forth as CACAGCAACTACCCCTGCTCCCACTAA (SEQ ID NO: 41). In several embodiments, a probe capable of hybridizing to a Ureaplasma urealyticum mba nucleic acid molecule consists essentially of, or consists of, a nucleic acid sequence set forth as SEQ ID NO: 41.

In several embodiments, a probe capable of hybridizing to a Ureaplasma spp. ure nucleic molecule contains a nucleic acid sequence that is at least 90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% identical, to the nucleotide sequence set forth as CCACCAGCAATAACAGTTGTAATACCACCATC (SEQ ID NO: 47). In several embodiments, a probe capable of hybridizing to a Ureaplasma spp. ure nucleic acid molecule consists essentially of, or consists of, a nucleic acid sequence set forth as SEQ ID NO: 47. In several examples, a probe capable of hybridizing to a Ureaplasma spp. ure nucleic acid molecule includes or consists of a nucleic acid sequence set forth as CCACCAGCAA“T”AACAGTTGTAATACCACCATC (SEQ ID NO: 47), where “T” is BHQ1. In particular embodiments, the probe capable of hybridizing to a Ureaplasma spp. ure nucleic acid molecule is capable of hybridizing to a ure nucleic acid molecule from any Ureaplasma species or serogroup (for example, Ureaplasma parvum or Ureaplasma urealyticum).

In additional embodiments, a probe is capable of hybridizing under high or very high stringency conditions to a Bartonella spp. nucleic acid sequence set forth as SEQ ID NO: 52. In several embodiments, a probe capable of hybridizing to a Bartonella spp. ssrA nucleic molecule contains a nucleic acid sequence that is at least 90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% identical, to the nucleotide sequence set forth as ACCCCGCTTAAACCTGCGACG (SEQ ID NO: 50). In several embodiments, a probe capable of hybridizing to a Bartonella spp. ssrA nucleic acid molecule consists essentially of, or consists of, a nucleic acid sequence set forth as SEQ ID NO: 50. In particular embodiments, the probe capable of hybridizing to a Bartonella spp. ssrA nucleic acid molecule is capable of hybridizing to a ssrA nucleic acid molecule from any Bartonella species or serogroup (for example, B. alsatica, B. bacilliformis, B. birtlesii, B. bovis, B capreoli, B. chomelii, B. clarridgeiae, B. doshiae, B. elizabethae, B. henselae, B. grahamii, B. japonica, B. koehlerae, B. melophagi, B. phoceensis, B. quintana, B. rochalimae, B. schoenbuchensis, B. silvatica, B. taeniae, B. taylorii, B. tribocorum, B. vinsonii, and/or B. washoensis).

In some embodiments, a probe is capable of hybridizing under high or very high stringency conditions to a Streptococcus agalactiae nucleic acid sequence set forth as SEQ ID NO: 53. In some examples, a probe capable of hybridizing to a Streptococcus agalactiae cfb nucleic molecule includes a nucleic acid sequence that is at least 90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleotide sequence set forth as AGACTTCATTGCGTGCCAACCCTGAGAC (SEQ ID NO: 56). In several embodiments, a probe capable of hybridizing to a Streptococcus agalactiae cfb nucleic acid molecule consists essentially of, or consists of, a nucleic acid sequence set forth as SEQ ID NO: 56.

In further embodiments, a probe is capable of hybridizing under high or very high stringency conditions to a Neisseria meningitidis nucleic acid sequence set forth as SEQ ID NO: 61. In some examples, a probe capable of hybridizing to a Neisseria meningitidis sodC nucleic molecule includes a nucleic acid sequence that is at least 90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleotide sequence set forth as CGCAGGCGGTCACTGGGATC (SEQ ID NO: 64). In several embodiments, a probe capable of hybridizing to a Neisseria meningitidis sodC nucleic acid molecule consists essentially of, or consists of, a nucleic acid sequence set forth as SEQ ID NO: 64.

In some examples, the probe is labeled with one or more fluorophores. Examples of suitable fluorophore labels are provided above. In some examples, the fluorophore is a donor fluorophore. In particular, non-limiting examples, the probes disclosed herein are labeled with FAM, although one of ordinary skill in the art can select other fluorophore labels for use in the disclosed methods. In other examples, the fluorophore is an accepter fluorophore, such as a fluorescence quencher. In some examples, the probe includes both a donor fluorophore and an accepter or quencher fluorophore, for example a donor fluorophore such as a FAM and an acceptor fluorophore such as a BLACK HOLE® quencher (such as BHQ1, BHQ2, or BHQ3) or TAMRA. Appropriate donor/acceptor fluorophore pairs can be selected using routine methods. In one example, the donor emission wavelength is one that can significantly excite the acceptor, thereby generating a detectable emission from the acceptor. In some examples, the probe is modified at the 3′-end to prevent extension of the probe by a polymerase.

In some examples, the acceptor fluorophore (such as a fluorescence quencher) is attached to the 3′ end of the probe and the donor fluorophore is attached to a 5′ end of the probe. In other examples, the acceptor fluorophore (such as a fluorescence quencher) is attached to the 5′ end of the probe and the donor fluorophore is attached to a 3′ end of the probe. In another particular example, the acceptor fluorophore (such as a fluorescence quencher) is attached to a modified nucleotide (such as a T) and the donor fluorophore is attached to a 5′ end of the probe. In some examples, the donor fluorophore is FAM and the acceptor fluorophore is BHQ1. In particular embodiments, the probes disclosed herein include a donor fluorophore attached to the 5′ end and an acceptor fluorophore attached to the 3′ end.

B. Primers

Primers capable of hybridizing to and directing the amplification of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcus agalactiae, or Neisseria meningitidis nucleic acid molecules are also disclosed. The primers disclosed herein are between 10 to 40 nucleotides in length, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or even 40 nucleotides in length. In some examples, the primers are at least 10, 15, 20, 25, 30, 35, or 40 nucleotides in length. In other examples, the primers may be no more than 10, 15, 20, 25, 30, 35, or 40 nucleotides in length.

In several embodiments, a primer is capable of hybridizing to and directing the amplification of an Acinetobacter baumannii oxa-51 nucleic acid molecule (such as SEQ ID NO: 1) and includes a nucleic acid sequence that is at least 90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleic acid sequence set forth as TATTTTTATTTCAGCCTGCTCACCTT (SEQ ID NO: 12) or AAATACTTCTGTGGTGGTTGCCTTA (SEQ ID NO: 13). In several embodiments, a primer capable of hybridizing to and directing the amplification of an Acinetobacter baumannii oxa-51 nucleic acid molecule consists essentially of, or consists of a nucleic acid sequence set forth as SEQ ID NO: 12 or SEQ ID NO: 13.

In some embodiments, a primer is capable of hybridizing to and directing the amplification of a Chlamydia trachomatis tmRNA nucleic acid molecule (such as SEQ ID NO: 2) and includes a nucleic acid sequence that is at least 90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleic acid sequence set forth as GGTGTAAAGGTTTCGACTTAGAA (SEQ ID NO: 36) or CGAACACCGGGTCACC (SEQ ID NO: 37). In several embodiments, a primer capable of hybridizing to and directing the amplification of a Chlamydia trachomatis tmRNA nucleic acid molecule consists essentially of, or consists of a nucleic acid sequence set forth as SEQ ID NO: 36 or SEQ ID NO: 37.

In other embodiments, a primer is capable of hybridizing to and directing the amplification of an Escherichia coli and/or Shigella spp. nucleic acid molecule (such as SEQ ID NO: 3) and includes a nucleic acid sequence that is at least 90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleic acid sequence set forth as GAGCATCAGGGTGGCTATACG (SEQ ID NO: 27) or ATAGTCTGCCAGTTCAGTTC (SEQ ID NO: 28). In several embodiments, a primer capable of hybridizing to and directing the amplification of an Escherichia coli and/or Shigella spp. nucleic acid molecule consists essentially of, or consists of a nucleic acid sequence set forth as SEQ ID NO: 27 or SEQ ID NO: 28.

In additional embodiments, a primer is capable of hybridizing to and directing the amplification of a Klebsiella pneumoniae nifA nucleic acid molecule (such as SEQ ID NO: 4) and includes a nucleic acid sequence that is at least 90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleic acid sequence set forth as TGCTGCATAAAGGCATCGTT (SEQ ID NO: 18) or CCACCGAGGCCAGCAA (SEQ ID NO: 19). In several embodiments, a primer capable of hybridizing to and directing the amplification of a Klebsiella pneumoniae nifA nucleic acid molecule consists essentially of, or consists of a nucleic acid sequence set forth as SEQ ID NO: 18 or SEQ ID NO: 19. In further embodiments, a primer is capable of hybridizing to and directing the amplification of a Klebsiella pneumoniae diguanylate cyclase nucleic acid molecule (such as SEQ ID NO: 57) and includes a nucleic acid sequence that is at least 90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleic acid sequence set forth as TGCAGATAATTCACGCCCAG (SEQ ID NO: 58) or ACCCGCTGGACGCCAT (SEQ ID NO: 59). In several embodiments, a primer capable of hybridizing to and directing the amplification of a Klebsiella pneumoniae diguanylate cyclase nucleic acid molecule consists essentially of, or consists of a nucleic acid sequence set forth as SEQ ID NO: 58 or SEQ ID NO: 59.

In further embodiments, a primer is capable of hybridizing to and directing the amplification of a Moraxella catarrhalis purH nucleic acid molecule (such as SEQ ID NO: 5) and includes a nucleic acid sequence that is at least 90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleic acid sequence set forth as GGTGAGTTGCCACAGC (SEQ ID NO: 24) or AGTAGACCGCCATTGACTC (SEQ ID NO: 25). In several embodiments, a primer capable of hybridizing to and directing the amplification of a Moraxella catarrhalis purH nucleic acid molecule consists essentially of, or consists of a nucleic acid sequence set forth as SEQ ID NO: 24 or SEQ ID NO: 25.

In still further embodiments, a primer is capable of hybridizing to and directing the amplification of a Pneumocystis jirovecii dhps nucleic acid molecule (such as SEQ ID NO: 6) and includes a nucleic acid sequence that is at least 90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleic acid sequence set forth as TAATGGTTTGCCTTGGTTGCTT (SEQ ID NO: 33) or CACAGCCTCCTAAAACAGAT (SEQ ID NO: 34). In several embodiments, a primer capable of hybridizing to and directing the amplification of a Pneumocystis jirovecii dhps nucleic acid molecule consists essentially of, or consists of a nucleic acid sequence set forth as SEQ ID NO: 33 or SEQ ID NO: 34.

In other embodiments, a primer is capable of hybridizing to and directing the amplification of a Pseudomonas aeruginosa gyrB nucleic acid molecule (such as SEQ ID NO: 7) and includes a nucleic acid sequence that is at least 90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleic acid sequence set forth as GTCTCGGTGGTGAACG (SEQ ID NO: 15) or TGGATGTTGCTGAAGGTCTC (SEQ ID NO: 16). In several embodiments, a primer capable of hybridizing to and directing the amplification of a Pseudomonas aeruginosa gyrB nucleic acid molecule consists essentially of, or consists of a nucleic acid sequence set forth as SEQ ID NO: 15 or SEQ ID NO: 16.

In additional embodiments, a primer is capable of hybridizing to and directing the amplification of a Staphylococcus aureus gsf nucleic acid molecule (such as SEQ ID NO: 8) and includes a nucleic acid sequence that is at least 90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleic acid sequence set forth as CGGGTTAGGTGAATTGATTGTTTTAT (SEQ ID NO: 30) or CGCATTTGAGCTGAAGTTG (SEQ ID NO: 31). In several embodiments, a primer capable of hybridizing to and directing the amplification of a Staphylococcus aureus gsf nucleic acid molecule consists essentially of, or consists of a nucleic acid sequence set forth as SEQ ID NO: 30 or SEQ ID NO: 31.

In other embodiments, a primer is capable of hybridizing to and directing the amplification of a Toxoplasma gondii ssrRNA nucleic acid molecule (such as SEQ ID NO: 9) and includes a nucleic acid sequence that is at least 90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleic acid sequence set forth as GGTGGTCCTCAGGTGAT (SEQ ID NO: 21) or CCACGGTAGTCCAATACAGTA (SEQ ID NO: 22). In several embodiments, a primer capable of hybridizing to and directing the amplification of a Toxoplasma gondii ssrRNA nucleic acid molecule consists essentially of, or consists of a nucleic acid sequence set forth as SEQ ID NO: 21 or SEQ ID NO: 22.

In further embodiments, a primer is capable of hybridizing to and directing the amplification of a Ureaplasma parvum ure nucleic acid molecule (such as SEQ ID NO: 10) and includes a nucleic acid sequence that is at least 90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleic acid sequence set forth as ACAGATAATGTTGATATGATTGTGGGTAT (SEQ ID NO: 42) or CTAATGCAACAGGAACTATTTCTG (SEQ ID NO: 43). In several embodiments, a primer capable of hybridizing to and directing the amplification of a Ureaplasma parvum ure nucleic acid molecule consists essentially of, or consists of a nucleic acid sequence set forth as SEQ ID NO: 42 or SEQ ID NO: 43.

In still further embodiments, a primer is capable of hybridizing to and directing the amplification of a Ureaplasma urealyticum mba nucleic acid molecule (such as SEQ ID NO: 11) and includes a nucleic acid sequence that is at least 90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleic acid sequence set forth as ATTTCATATTTAGTTTATTAGGAGATCGTTAT (SEQ ID NO: 39) or AGATTTAACATTTGAGCTAGAACAT (SEQ ID NO: 40). In several embodiments, a primer capable of hybridizing to and directing the amplification of a Ureaplasma urealyticum mba nucleic acid molecule consists essentially of, or consists of a nucleic acid sequence set forth as SEQ ID NO: 39 or SEQ ID NO: 40.

In additional embodiments, a primer is capable of hybridizing to and directing the amplification of a Ureaplasma spp. ure nucleic acid molecule (such as SEQ ID NO: 10) and includes a nucleic acid sequence that is at least 90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleic acid sequence set forth as GGTTTAGATACTCACGTTCACTGA (SEQ ID NO: 45) or GCTTTTGTACCATCATTCATACCTGT (SEQ ID NO: 46). In several embodiments, a primer capable of hybridizing to and directing the amplification of a Ureaplasma spp. ure nucleic acid molecule consists essentially of, or consists of a nucleic acid sequence set forth as SEQ ID NO: 45 or SEQ ID NO: 46.

In additional embodiments, a primer is capable of hybridizing to and directing the amplification of a Bartonella spp. ssrA nucleic acid molecule (such as SEQ ID NO: 52) and includes a nucleic acid sequence that is at least 90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleic acid sequence set forth as GCTATGGTAATAAATGGACAATGAAATAA (SEQ ID NO: 48), GCTTCTGTTGCCAGGTG (SEQ ID NO: 49), or CTAAATGAGTAGTTGCAAATGACAAC (SEQ ID NO: 51). In several embodiments, a primer capable of hybridizing to and directing the amplification of a Bartonella spp. ssrA nucleic acid molecule consists essentially of, or consists of a nucleic acid sequence set forth as SEQ ID NO: 48, SEQ ID NO: 49, or SEQ ID NO: 51. In particular embodiments, the primer capable of hybridizing to and directing amplification of a Bartonella spp. ssrA nucleic acid molecule is capable of hybridizing to a ssrA nucleic acid molecule from any Bartonella species or serogroup (for example, B. alsatica, B. bacilliformis, B. birtlesii, B. bovis, B capreoli, B. chomelii, B. clarridgeiae, B. doshiae, B. elizabethae, B. henselae, B. grahamii, B. japonica, B. koehlerae, B. melophagi, B. phoceensis, B. quintana, B. rochalimae, B. schoenbuchensis, B. silvatica, B. taeniae, B. taylorii, B. tribocorum, B. vinsonii, and/or B. washoensis).

In additional embodiments, a primer is capable of hybridizing to and directing the amplification of a Streptococcus agalactiae cfb nucleic acid molecule (such as SEQ ID NO: 53) and includes a nucleic acid sequence that is at least 90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleic acid sequence set forth as GGGAACAGATTATGAAAAACCG (SEQ ID NO: 54) or AAGGCTTCTACACGACTACCAA (SEQ ID NO: 55). In several embodiments, a primer capable of hybridizing to and directing the amplification of a Streptococcus agalactiae cfb nucleic acid molecule consists essentially of, or consists of a nucleic acid sequence set forth as SEQ ID NO: 54 or SEQ ID NO: 55.

In additional embodiments, a primer is capable of hybridizing to and directing the amplification of a Neisseria meningitidis sodC nucleic acid molecule (such as SEQ ID NO: 61) and includes a nucleic acid sequence that is at least 90% identical, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical, to the nucleic acid sequence set forth as CTGTGAGCCAAAAGAAAAAGAAG (SEQ ID NO: 62) or GATTTGTTGCTGTGCCATCAT (SEQ ID NO: 63). In several embodiments, a primer capable of hybridizing to and directing the amplification of a Neisseria meningitidis sodC nucleic acid molecule consists essentially of, or consists of a nucleic acid sequence set forth as SEQ ID NO: 62 or SEQ ID NO: 63.

In certain embodiments, the primers are utilized or provided as a set of primers, such as a pair of primers, capable of hybridizing to and amplifying a disclosed nucleic acid, such as one of SEQ ID NOs: 1-11, 52-53, 57, or 60. In some examples, the set of primers includes a pair of primers including SEQ ID NOs: 12 and 13, a pair of primers including SEQ ID NOs: 15 and 16, a pair of primers including SEQ ID NOs: 18 and 19, a pair of primers including SEQ ID NOs: 21 and 22, a pair of primers including SEQ ID NOs: 24 and 25, a pair of primers including SEQ ID NOs: 27 and 28, a pair of primers including SEQ ID NOs: 30 and 31, a pair of primers including SEQ ID NOs: 33 and 34, a pair of primers including SEQ ID NOs: 36 and 37, a pair of primers including SEQ ID NOs: 39 and 40, a pair of primers including SEQ ID NOs: 42 and 43, a pair of primers including SEQ ID NOs: 45 and 46, a pair of primers including SEQ ID NOs: 48 and 49, a pair of primers including SEQ ID NOs: 51 and 49, a pair of primers including SEQ ID NO: 54 and SEQ ID NO: 55, a pair of primers including SEQ ID NO: 58 and SEQ ID NO: 59, or a pair of primers including SEQ ID NO: 62 and SEQ IDNO: 63.

C. Probe and Primer Variants

Although exemplary probe and primer sequences are provided in SEQ ID NOs: 12-51, 54-56, 58-60, and 62-64, the primer and/or probe sequences can be varied slightly by moving the probe or primer a few nucleotides upstream or downstream from the nucleotide positions that they hybridize to on the target nucleic molecule acid, provided that the probe and/or primer is still specific for the target nucleic acid sequence, for example specific for one of SEQ ID NOs: 1-11, 52-53, 57, or 60. For example, variations of the probes and primers disclosed as SEQ ID NOs: 12-51, 54-56, 58-60, and 62-64 can be made by “sliding” the probes and/or primers a few nucleotides 5′ or 3′ from their positions, and such variation will still be specific for the respective target nucleic acid sequence.

Also provided by the present disclosure are probes and primers that include variations to the nucleotide sequences shown in any of SEQ ID NOs: 12-51, 54-56, 58-60, and 62-64, as long as such variations permit detection of the target nucleic acid molecule. For example, a probe or primer can have at least 90% sequence identity such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a nucleic acid including of the sequence shown in any of SEQ ID NOs: 12-51, 54-56, 58-60, and 62-64. In such examples, the number of nucleotides does not change, but the nucleic acid sequence shown in any of SEQ ID NOs: 12-51, 54-56, 58-60, and 62-64 can vary at a few nucleotides, such as changes at 1, 2, 3, or 4 nucleotides.

The present application also provides probes and primers that are slightly longer or shorter than the nucleotide sequences shown in any of SEQ ID NOs: 12-51, 54-56, 58-60, and 62-64, as long as such deletions or additions permit detection of the desired target nucleic acid molecule, such as one of SEQ ID NOs: 1-11, 52-53, 57, or 60. For example, a probe or primer can include a few nucleotide deletions or additions at the 5′- or 3′-end of the probe or primers shown in any of SEQ ID NOs: 12-51, 54-56, 58-60, and 62-64, such as addition or deletion of 1, 2, 3, or 4 nucleotides from the 5′- or 3′-end, or combinations thereof (such as a deletion from one end and an addition to the other end). In such examples, the number of nucleotides changes.

Also provided are probes and primers that are degenerate at one or more positions (such as 1, 2, 3, 4, 5, or more positions), for example, a probe or primer that includes a mixture of nucleotides (such as 2, 3, or 4 nucleotides) at a specified position in the probe or primer. In some examples, the probes and primers disclosed herein include one or more synthetic bases or alternative bases (such as inosine). In other examples, the probes and primers disclosed herein include one or more modified nucleotides or nucleic acid analogues, such as one or more locked nucleic acids (see, e.g., U.S. Pat. No. 6,794,499) or one or more superbases (Nanogen, Inc., Bothell, Wash.). In other examples, the probes and primers disclosed herein include a minor groove binder conjugated to the 5′ or 3′ end of the oligonucleotide (see, e.g., U.S. Pat. No. 6,486,308).

V. Kits

The nucleic acid primers and probes disclosed herein can be supplied in the form of a kit for use in the detection of one or more of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumooniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcus agalactiae, or Neisseria meningitidis in a sample. In such a kit, an appropriate amount of one or more of the nucleic acid probes and/or primers (such as one or more of SEQ ID NOs: 12-51, 54-56, 58-60, and 62-64) are provided in one or more containers or in one or more individual wells of a multiwall plate or card. A nucleic acid probe and/or primer may be provided suspended in an aqueous solution or as a freeze-dried or lyophilized powder, for instance. The container(s) in which the nucleic acid(s) are supplied can be any conventional container that is capable of holding the supplied form, for instance, microfuge tubes, ampoules, or bottles. The kits can include either labeled or unlabeled nucleic acid probes (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 probes) for use in detection of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcus agalactiae, or Neisseria meningitidis nucleic acids. One or more control probes and/or primers for use in the PCR reactions also may be supplied in the kit. In some examples, the probes are detectably labeled.

In some examples, one or more sets of primers (such as the primers described above), such as pairs of primers (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 pairs of primers), may be provided in pre-measured single use amounts in individual, typically disposable, tubes, wells, or equivalent containers. With such an arrangement, the sample to be tested for the presence of the target nucleic acids can be added to the individual tube(s) or well(s) and amplification carried out directly.

The amount of nucleic acid primer supplied in the kit can be any appropriate amount, and may depend on the target market to which the product is directed. For instance, if the kit is adapted for research or clinical use, the amount of each nucleic acid primer provided would likely be an amount sufficient to prime several PCR amplification reactions. General guidelines for determining appropriate amounts may be found in Innis et al., Sambrook et al., and Ausubel et al. A kit may include more than two primers in order to facilitate the PCR amplification of a larger number of target nucleic acid molecules, such as Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumooniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli/Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcus agalactiae, or Neisseria meningitidis nucleic acids, or any combination of two or more thereof.

In some embodiments, kits also may include the reagents necessary to carry out PCR amplification reactions, including DNA sample preparation reagents, appropriate buffers (such as polymerase buffer), salts (for example, magnesium chloride), deoxyribonucleotides (dNTPs), and polymerases.

In particular embodiments, the kits include prepackaged probes, such as probes suspended in suitable medium in individual containers (for example, individually sealed tubes or wells). In some examples, the probes include those provided herein. In other particular embodiments, the kit includes equipment, reagents, and instructions for extracting and/or purifying nucleotides from a sample.

The present disclosure is illustrated by the following non-limiting Examples.

EXAMPLE 1 Primers and Probes

Primers and probes were designed for detection of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumooniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli/Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp., and Streptococcus agalactiae. In most cases, oligonucleotides were designed using Primer Express 3.0 software (Applied Biosystems, Foster City, Calif.) with slight modifications to optimize melting temperatures (Tm) and minimize intra- and inter-molecular interactions. Specificity of each set of oligonucleotides for the intended genus and/or species was assessed by sequence comparison using Basic Local Alignment Search Tool (BLAST) within the National Center for Biotechnology Information (NCBI) database (available on the world wide web at blast.ncbi.nlm.nih.gov/Blast.cgi). Primers and hydrolysis probes used for analytical validation were manufactured by the Biotechnology Core Facility at the Centers for Disease Control and Prevention (Atlanta, Ga., USA).

The target genes and primer and probe sequences are shown in Table 1. Some probes (such as the P. jirovecii, U. parvum, and Ureaplasma spp. probes) include a BHQ1 internal quencher linked to a “T” nucleotide. Probes including an internal quencher include C6 CpG at the 3′ end to prevent extension. In some examples, each probe includes 5′ FAM label and a 3′ BHQ1 label, except where indicated otherwise.

TABLE 1 Primers and probes for detection of pathogens SEQ Final ID Conc. Organism Target Oligo Sequence NO: (nM) Acinetobacter oxa-51 AB F1 TATTTTTATTTCAGCCTG 12 1000 baumannii CTCACCTT AB R3 AAATACTTCTGTGGTGGT 13 1000 TGCCTTA AB P1 TGACTGCTAATCCAAATC 14 200 ACAGCGCTTCA Pseudomonas gyrB PA F2 GTCTCGGTGGTGAACG 15 500 aeruginosa PA R TGGATGTTGCTGAAGGTC 16 500 TC PA P2 TCCGTCGCCACAACAAGG 17 100 TCTGGGAA Klebsiella nifA KP F1 TGCTGCATAAAGGCATCG 18 1000 pneumoniae TT KP R1 CCACCGAGGCCAGCAA 19 1000 KP P1 ACGCTGAGCACCTCCTGC 20 200 AACGT diguanylate For TGCAGATAATTCACGCCC 58 1000 cyclase AG Rev ACCCGCTGGACGCCAT 59 1000 Probe CCACCACGCTCATCTGTT 60 200 TCGCC Toxoplasma ssrRNA TG F2 GGTGGTCCTCAGGTGAT 21 1000 gondii TG R2 CCACGGTAGTCCAATACA 22 1000 GTA TG P2 ATCGCGTTGACTTCGGTC 23 200 TGCGAC Moraxella purH MC F2 GGTGAGTTGCCACAGC 24 1000 catarrhalis MC R2 AGTAGACCGCCATTGACT 25 1000 C MC P1 CACAGCGGGCAGCTCAAT 26 200 TTGACCTA Escherichia uidA ECS F1A GAGCATCAGGGTGGCTAT 27 500 coli/ ACG Shigella ECS R1A ATAGTCTGCCAGTTCAGT 28 500 TC ECS P1 TACGGCGTGACATCGGCT 29 100 TCAAATG Staphylococcus gsf SA F2 CGGGTTAGGTGAATTGAT 30 1000 aureus TGTTTTAT SA R2 CGCATTTGAGCTGAAGTT 31 1000 G SA P2 TTCCATATGACCACCACG 32 200 AGTCTTAGCACC Pneumocystis dhps PJ F1 TAATGGTTTGCCTTGGTT 33 1000 jirovecii GCTT PJ R2A CACAGCCTCCTAAAACAG 34 1000 AT PJ P2A ACAGGGTGTCT“T”ACAG 35 200 GTGATGTTATGCCAAAAG Chlamydia tmRNA CT F1 GGTGTAAAGGTTTCGACT 36 1000 trachomatis TAGAA CT R3 CGAACACCGGGTCACC 37 1000 CT P1 ATGCGGAGGGCGTTGGCT 38 200 GG Ureaplasma mba UU F1 ATTTCATATTTAGTTTAT 39 1000 urealyticum TAGGAGATCGTTAT UU R1 AGATTTAACATTTGAGCT 40 1000 AGAACAT UU P2 CACAGCAACTACCCCTGC 41 200 TCCCACTAA Ureaplasma ure PARVUM F3 ACAGATAATGTTGATATG 42 1000 parvum ATTGTGGGTAT PARVUM R3 CTAATGCAACAGGAACTA 43 1000 TTTCTG PARVUM P3i TCAGTGAACG“T”GAGTA 44 200 TCTAAACCACCAGC Ureaplasma ure UPAN F1 GGTTTAGATACTCACGTT 45 500 spp. CACTGA UPAN R1 GCTTTTGTACCATCATTC 46 500 ATACCTGT UPAN P1i CCACCAGCAA“T”AACAG 47 100 TTGTAATACCACCATC Bartonella ssrA ssrA- GCTATGGTAATAAATGGA 48 500 spp. F CAATGAAATAA ssrA- GCTTCTGTTGCCAGGTG 49 500 R ssrA- ACCCCGCTTAAACCTGCG 50 100 P ACG ssrA- CTAAATGAGTAGTTGCAA 51 F2 ATGACAAC Streptococcus cfb For GGGAACAGATTATGAAAA 54 1000 agalactiae ACCG Rev AAGGCTTCTACACGACTA 55 1000 CCAA Probe AGACTTCATTGCGTGCCA 56 200 ACCCTGAGAC Neisseria sodC For CTGTGAGCCAAAAGAAAA 62 1000 meningitidis AGAAG Rev GATTTGTTGCTGTGCCAT 63 1000 CAT Probe CGCAGGCGGTCACTGGGA 64 200 TC Salmonella ttrRSBCA For CTCACCAGGAGATTACAA 65 500 spp. CATGG Rev AGCTCAGACCAAAAGTGA 66 500 CCATC Probe CACCGACGGCGAGACCGA 67 100 CTTT “T” = BHQ1 modification

EXAMPLE 2 Detection and Differentiation of Bartonella Species and Genotypes

Bacterial strains and DNA extraction: All bacterial strains were obtained from collections at the Centers for Disease Control and Prevention in Fort Collins, CO and Atlanta, Ga. Nucleic acid was extracted from 33 Bartonella strains, including 25 defined species or subspecies using the QIAAMP DNA mini kit (Qiagen, Valencia, Calif.). Bartonella strains included in this study were: B. alsatica (IBS 382), B. bacilliformis (KC584), B. birtlesii (IBS 325), B. bovis (91-4), B. capreoli (WY-Elk), B. chomelii (A828), B. clarridgeiae (Houston-2), B. doshiae (R18), B. elizabethae (F9251), B. henselae (Houston-1), B. grahamii (V2), B. japonica (Fuji 18-1T), B. koehlerae (C-29), B. melophagi (K-2C), B. phoceensis (16120), B. quintana (Fuller), B. rochalimae (BMGH), B. schoenbuchensis (R1), B. silvatica (Fuji 23-1T), B. tamiae (Th307, Th239, and Th339), B. taylorii (M16), B. tribocorum (IBS 506), B. vinsonii subsp. arupensis (OK 94-513), B. vinsonii subsp. vinsonii (Baker), B. washoensis (Sb944nv), and Bartonella isolates (Sh6397ga, Sh6396ga, Sh6537ga, Sh8784ga, Sh8200ga, and Sh8776ga). Using the MAGNA PURE® Compact instrument with Total Nucleic Acid Isolation Kit I (Roche Applied Science, Indianapolis, Ind.), nucleic acid was extracted from 61 microorganisms that are closely related genetically to Bartonella or may occupy a similar ecological niche, including Afipia broomii, Afipia clevelandensis, Afipia felis, Agrobacterium radiobacter, Agrobacterium tumefaciens, Babesia microti, Bordetella pertussis, Bordetella parapertussis, Bradyrhizobium, Brucella abortus, Brucella canis, Brucella melitensis, Brucella neotomae, Brucella ovis, Brucella suis, Campylobacter coli, Campylobacter fetus, Campylobacter jejuni, Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae, Erwinia, Escherichia albertii (2 strains), Escherichia blattae, Escherichia coli (4 strains), Escherichia fergusonii, Escherichia hermanii, Escherichia vulneris, Haemophilus influenzae, Klebsiella oxytoca, Klebsiella pneumoniae, Kluyvera intermedia, Legionella pneumophila, Methylobacterium organophilum, Ochrobactrum anthropi (3 strains), Ochrobactrum intermedium, Oligella urethralis (4 strains), Psychrobacter phenylpyruvicus (2 strains), Raoultella planticola, Salmonella bongori, Salmonella enterica (serovar Enteriditis, serovar Typhi, serovar Typhimurium), Shigella boydii, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Toxoplasma gondii, and Vibrio cholerae. Human genomic DNA was also tested for cross-reactivity. All nucleic acid extracts were normalized to 1 ng/μL in Tris-EDTA buffer.

Real-time PCR: Sequences of the ssrA (tmRNA) gene of five representative Bartonella species were obtained from the tmRNA Website (indiana.edu/˜tmrna/) and GenBank (accession numbers: NC_005955.1, NC_005956.1, NC_010161.1, NC_012846.1, NC_008783.1). Sequences were aligned using the Clustal W method (ebi.ac.uk/Tools/msa/clustalw2/). Primers and probes were designed using Primer Express 3.0 software (Applied Biosystems, Foster City, Calif.) with some modification for amplification of a 301 bp region of ssrA. The reaction mix (25 μL) contained the following components: 12.5 μL 2× PerfeCta® MultiPlex qPCR SuperMix (Quanta Biosciences, Gaithersburg, Md.), forward and reverse primers (ssrA-F: 5′-GCTATGGTAATAAATGGACAATGAAATAA-3′ (SEQ ID NO: 48), ssrA-R: 5′-GCTTCTGTTGCCAGGTG-3′ (SEQ ID NO: 49)) at a final concentration of 500 nM, FAM-labeled probe (5′FAM-ACCCCGCTTAAACCTGCGACG-3′BHQ1 (SEQ ID NO: 50)) at a final concentration of 100 nM, and 5 μL of extracted nucleic acid. Real-time PCR was performed on the Applied Biosystems 7500 real-time PCR instrument with the following thermocycling parameters: 1 cycle of 95° C. for 2 min followed by 45 cycles of 95° C. for 15 sec and 60° C. for 60 sec with data collection in the FAM channel. Primers and probe were tested using nuclease-free water (n=95) to ensure no signal in the absence of nucleic acid template. The limit of detection was independently determined and verified for four species (B. quintana, B. henselae, B. bovis, and B. elizabethae) by testing 10 replicates each of 10-fold serial dilutions of genomic DNA ranging from 1 ng/μL to 0.1 fg/μL. The limit of detection was identified as the lowest dilution at which amplification was observed in at least 50% of replicates. Specificity was assessed by performing the assay using 15 ng of nucleic acid from 61 different microorganisms representing 24 genera and 48 species.

Sequencing: Amplicons for sequencing were generated by conventional PCR with forward and reverse primers at 400 nM each using the Bio-Rad Dyad® thermal cycler (Bio-Rad, Hercules, Calif.) with the following thermocycling conditions: 95° for 2 min, 30 cycles of 95° for 15 sec, 60° for 60 sec, and 72° for 30 sec followed by 72° for 3 min. Amplicons were visualized by electrophoresis in a 1% agarose gel followed by staining with 0.05% methylene blue solution and purification using the Geneclean® Turbo kit (MP Biomedicals, Solon, Ohio). Sequencing reactions were performed in both directions using BigDye® Terminator 3.1 cycle sequencing kit (Applied Biosystems) according to the manufacturer's instructions with the same primers for the real-time PCR assay at a final concentration of 165 nM. Sequencing was performed on the Applied Biosystems 3130×L genetic analyzer.

Phylogenetic analysis: A 253 bp region of each amplified sequence (excluding forward and reverse primers) was used for alignment and phylogenetic comparison of Bartonella species and genotypes using Lasergene® version 8 software suite (DNASTAR, Madison, Wis.). All ssrA sequences were aligned using the Clustal V method. Phylogenetic trees were constructed using the neighbor-joining method and bootstrapping analysis with 1,000 replicates.

Testing of animal blood: Blood specimens collected from elk (Cervus elaphus) in Wyoming (n=56) and cattle (Bos primigenius) in the country of Georgia (n=89) between 2008-2009 were tested for Bartonella by bacterial culture using previously described methods (Bai et al., Vet. Microbiol. 148:329-332, 2011). The culture results from this cohort of elk have been reported previously (Bai et al., 2011). All specimens were extracted using the DNEASY® Blood and Tissue kit (Qiagen) or MAGNA PURE® Compact with Total Nucleic Acid Isolation Kit I (Roche Applied Science). Five or 10 μL of nucleic acid extract was used in each real-time PCR reaction.

Nucleotide sequence accession numbers: Thirty-four unique ssrA sequences obtained from Bartonella strains and isolates were submitted to GenBank and assigned the following accession numbers: JN029776 (B. alsatica IBS 382), JN029794 (B. bacilliformis KC584), JN029775 (B. birtlesii IBS325), JN029767 (B. bovis 91-4), JN029798 (B. capreoli WY-Elk), JN029773 (B. chomelii A828), JN029768 (B. doshiae R18), JN029774 (B. elizabethae F9251), JN029785 (B. henselae Houston-1), JN029795 (B. grahamii V2), JN029784 (B. japonica Fuji 18-1T), JN029769 (B. koehlerae C-29), JN029771 (B. melophagi K-2C), JN029770 (B. phoceensis 16120), JN029766 (B. quintana Fuller), JN029797 (B. rochalimae BMGH), JN029772 (B. schoenbuchensis R1), JN029782 (B. silvatica Fuji 23-1T), JN029778 (B. taeniae Th307), JN029779 (B. taeniae Th239), JN029780 (B. taeniae Th339), JN029781 (B. taylorii M16), JN029796 (B. tribocorum IBS 506), JN029783 (B. vinsonii subsp. arupensis OK 94-513), JN029777 (B. vinsonii subsp. vinsonii Baker), JN029786 (B. washoensis Sb944nv), JN029787 (Bartonella sp. Sh6397ga), JN029791 (Bartonella sp. Sh8200ga), JN029793 (Bartonella sp. Sh8776ga), JN029788 (Bartonella sp. Sh6396ga), JN029790 (Bartonella sp. Sh8784ga), JN029792 (Bartonella sp. Sh9282ga), JN029789 (Bartonella sp. Sh6537ga), JN982716 (B. clarridgeiae Houston-2). The ssrA sequence amplified from elk blood was assigned accession number JN982717, and the sequence identified in cattle blood was identical to B. bovis (JN029767).

Real-time PCR for detection of Bartonella ssrA: Amplification of the target sequence occurred with all Bartonella species (n=24) and unclassified Bartonella strains (n=7) tested (data not shown). Amplification curves demonstrated sigmoidal shape and had crossing threshold (Ct) values between 15 and 21 with 5 ng of DNA per reaction. No amplification was observed in no-template control (NTC) reactions (n=95) or with DNA from other microorganisms listed above (n=61) or human genomic DNA. The limit of detection was independently determined for four species (B. quintana, B. henselae, B. bovis, and B. elizabethae) and found to be ≤5 fg of nucleic acid per reaction.

Bartonella phylogeny based on ssrA genotypes: Phylogenetic analysis of ssrA sequences from each Bartonella strain or isolate showed that this region was sufficient to discriminate all Bartonella species and that separation of clades based on ssrA sequences was consistent with phylogeny based on gltA, which is considered a reliable tool for distinguishing closely related Bartonella genotypes (LaScola et al., Trends Microbiol. 11:318-321, 2003). First, the ssrA sequences from ruminant-associated Bartonella, including B. chomelii, 163 B. capreoli, B. bovis, B. melophagi, and B. schoenbuchensis formed an independent clade; sequence identity between these species was ≥94%. Further, both subspecies of B. vinsonii (vinsonii and arupensis) included in this study formed a separate grouping in the tree with 98% identity, as did three strains of the recently identified pathogenic Bartonella species B. taeniae (≥97.2% identity) (Kosoy et al., J. Clin. Microbiol. 46:772-775, 2008). Among all ssrA sequences, the lowest percent identity (75.3 to 84.1%) was observed for strains of B. taeniae relative to other Bartonella species, thus supporting the separation of B. taeniae as a novel species (Kosoy et al., 2008). The division of two additional clades which are similarly separated by gltA comparison, one consisting of B. elizabethae and B. tribocorum and the other including B. henselae and B. koehlerae, were also supported by the phylogenetic analysis of ssrA. Overall, the separation of major Bartonella clades based on ssrA sequences was consistent with phylogeny based on gltA (Kosoy et al., Am. J. Trop. Med. Hyg. 82:1140-1145, 2010; Maillard et al., Int. Syst. Evol. Microbiol. 54:215-220, 2004).

Detection and identification of Bartonella in animal blood: This assay was used to screen elk and cattle blood specimens for the presence of Bartonella and compared to bacterial culture results. Bartonella DNA was detected in 16 of 55 (29.1%) and 42 of 89 (47.2%) specimens from elk and cattle, respectively. The appropriate amplicon size was confirmed for positive samples. Using traditional culturing methods, Bartonella was recovered from only 4 of 55 (7.3%) elk and 34 of 89 (38.2%) cattle specimens. Since comparison of ssrA genotypes from Bartonella reference strains showed that this sequence provides sufficient information to discriminate Bartonella genotypes, we performed sequencing analysis of a subset of ssrA sequences amplified from elk (n=3) and cattle (n=5) specimens in order to identify the Bartonella species present. Analysis of ssrA sequences from elk blood revealed one genotype which clustered most closely with B. capreoli, a Bartonella species found in wild and domestic ruminants (Bai et al., Vet. Microbiol. 148:329-332, 2011). These results were consistent with previous identification of B. capreoli isolated from these samples using sequencing analysis of gltA (Bai et al., 2011). Similarly, a single ssrA genotype present in cattle blood was found to be identical to B. bovis (99.7% similarity). This result corroborated previous identification of B. bovis from these cattle specimens by analysis of gltA.

Samples from four patients in Thailand presenting with headache, myalgia, dizziness, fatigues, and rat exposure and animal ownership were analyzed for Bartonella infection. Blood clots from each patient were inoculated into Bartonella alpha-Proteobacteria growth medium and incubated aerobically at 35° C. with 5% CO₂ for seven days. DNA was extracted from this pre-enrichment using the QIAAMP® DNA mini kit (Qiagen, Chasworth, Calif.) according to manufacturer's instruction, and analyzed using real-time PCR targeting ssrA, as described above. PCR assays were performed using a CFX96™ Real-Time System (Bio-Rad, Hercules, Calif.). Amplicons were recovered from PCR reactions by gel-purification and sequenced in both directions using an Applied Biosystems Model 3130 Genetic Analyzer (Applied Biosystems, Foster City, Calif.).

Sequences obtained from the patients were very similar to the type strain of B. vinsonii subsp. arupensis. The ssrA sequences revealed two similar variants. One variant was identical to the type strain of B. vinsonii. subsp. arupensis (JN029783) and was identified in three of the patients (45-00250, 45-01217, and 45-01239). The other variant (JN394654), from patient 45-00025, was 2.8% divergent from the type strain of B. vinsonii. subsp. arupensis.

EXAMPLE 3 Multiple Pathogen Detection in Population-Based Study of Neonatal Infection Materials and Methods

Real-time PCR assay design and analytical validation: A panel of neonatologists with expertise in neonatal infection and South Asia was convened, and the Delphi method (Dalkey and Helmer, Management Sci. 9:458-467, 1963) was used to identify organisms of the highest priority for testing in nasopharyngeal (NP) and oropharyngeal (OP) swabs and blood specimens from neonates. Primers and hydrolysis probes were designed as described in Example 1.

All newly developed real-time PCR assays were evaluated using individual RT-qPCR reactions prior to use on the TAQMAN array card (TAC) format. Each assay was tested using nuclease-free water as template (n≥95) to ensure no fluorescence amplification signal was observed in the absence of nucleic acid. The limit of detection was independently determined for each assay by testing at least 3 replicates each of a 10-fold dilution series of specific total nucleic acid ranging from 0.1 fg/μL to 1 ng/μL. Inclusivity was assessed by testing representative isolates, including various subspecies, serotypes, or clonal groups, as appropriate (Table 2). Specificity of each assay was assessed by testing 15 ng of nucleic acid from at least 200 different bacteria, viruses, and protozoa representing 36 genera and 143 species. In addition to the most closely related species to each target pathogen, this panel also included commensals of the respiratory tract and human nucleic acid.

Oligonucleotide preparation for TAC manufacturing: Oligonucleotides for TAC production were manufactured by Integrated DNA Technologies (Coralville, Iowa) or Biosearch Technologies (Novato, Calif.), diluted and combined to 20× reaction concentration, and provided to Life Technologies (Foster City, Calif.) for custom manufacturing of study-specific TACs. The 20× concentration corresponds to a 1× final reaction concentration in the 1 μL reaction within each TAC well on the finished card. Total nucleic acid was extracted from a series of 10-fold dilutions of each organism and tested to determine the potential impact of oligonucleotide concentration on assay sensitivity.

Clinical specimens: Clinical specimens, including whole blood and combined NP/OP swabs, were obtained from enrolled neonates at three ANISA study sites: Sylhet, Bangladesh; Karachi, Pakistan; and Matiari, Pakistan. NP/OP swabs were collected and placed together in 1 mL Universal Transport Media (UTM, Copan Diagnostics, Murrieta, Calif.) and stored at −70° C. prior to extraction. Blood specimens were collected in standard EDTA collection tubes and stored at 4° C. for short-term storage (≤72 h post-collection) or −70° C. for longer storage prior to nucleic acid extraction and testing by TAC. Additional respiratory clinical specimens (NP/OP swabs) from the historical collection at the Centers for Disease Control and Prevention (Atlanta, Ga.) were also used for some experiments.

Specimen processing and nucleic acid extraction: Total nucleic acid (TNA) was extracted from clinical specimens using the MAGNA PURE® Compact instrument (Roche Applied Sciences, Indianapolis, Ind.) with Nucleic Acid Isolation Kit I and Total NA Plasma protocol. For NP/OP swab specimens, 400 μL of UTM was extracted and eluted in 100 μL. For extraction of whole blood, 300 μL of blood in EDTA was mixed with 100 μL of a freshly-prepared solution of lytic enzymes consisting of 1.5 mg/mL lysostaphin, 2500 U/mL mutanolysin, and 200 mg/mL lysozyme (Sigma-Aldrich, St. Louis, Mo.) in Tris-EDTA (TE) buffer and incubated at 37° C. for 30-60 min. prior to extraction on the MAGNA PURE® Compact, with elution in 100 To assess the potential impact of a pre-lysis step on recovery of TNA from a variety of pathogens, healthy donor blood was spiked with 10-fold serial dilutions of quantified culture stock of gram-positive bacteria (S. aureus), gram-negative bacterium (K. pneumoniae), or an RNA virus (enterovirus) and tested using individual RT-qPCR reactions. Spiked blood specimens were extracted directly or incubated with TE buffer or TE buffer containing lytic enzymes at 37° C. for 30 min. prior to extraction. Ct values for spiked blood experiments were compared using Student's two-tailed t test.

Individual real-time PCR assay performance: All individual real-time PCR assays were performed on the Applied Biosystems 7500 Real-Time PCR system (Life Technologies, Foster City, Calif.) with the following cycling conditions: 45° C. for 10 minutes, 94° C. for 10 minutes, 45 cycles of 94° C. for 30 seconds and 60° C. for 60 seconds, with data acquisition in the FAM channel during the 60° C. step. Each reaction consisted of 1× AGPATH-ID™ One-step RT-PCR buffer and 1× AGPATH-ID™ One-step RT-PCR enzyme mix (Applied Biosystems, Foster City, Calif.) or 1× QSCRIPT™ XLT One-step RT-qPCR TOUGHMIX®, low ROX (Quanta Biosciences, Gaithersburg, Md., USA), forward and reverse primers and FAM-labeled hydrolysis probe at the concentrations listed in Table 1, and nuclease-free water to final volume of 20 Five μL of TNA was used in each reaction.

TAC assay performance: Mastermix for each TAC consisted of the following: 1× AGPATH-ID™ One-step RT-PCR buffer and enzyme or 1× QSCRIPT™ XLT One-step RT-qPCR TOUGHMIX®. Reactions tested using AGPATH-ID™ enzyme system consisted of 50 μL 2× buffer, 4 μL 25× enzyme mix, and 46 μL of TNA. Reactions tested with the QSCRIPT™ enzyme system consisted of 50 μL 2× mastermix and 50 μL of TNA. Each card was centrifuged at 336×g for 1 min. twice, to distribute the fluid in the reaction wells, and sealed to sequester individual reactions. All TACs were run on the Applied Biosystems VIIA™ 7 Real-Time PCR system (Life Technologies, Foster City, Calif.) using the same cycling conditions as used for individual RT-qPCR reactions. A no template control (NTC) and a positive control consisting of combined RNA transcripts generated as previously described (Kodani and Winchell, J. Clin. Microbiol. 50:1057-1060, 2012) were included on each card.

Results

Analytical validation of new real-time PCR assays: For each new assay, no amplification was observed in no-template control (NTC) reactions (n≥95) or in reactions containing nucleic acid from other organisms (n≥200, data not shown). Each assay was also tested for inclusivity within the genus or species using representative isolates of each subspecies or serotype as appropriate (Table 2). The number of isolates used for inclusivity testing varied based on availability. The limit of detection was independently determined for each assay (Table 2).

TABLE 2 Analytical validation of newly developed real-time PCR assays for ANISA study No. of Limit of isolates Assay Detection tested Notes T. gondii <1 fg 1 S. aureus 100 fg-10 fg/μL 11 Tested representative isolates from various MRSA clonal groups (USA 100, 200, 300, 400, 500, 800, 1000, Brazilian, EMRSA 15, and ST80) K. pneumoniae 100 fg/μL 2 E. coli and 10 fg-1 fg/μL 94 Tested 21 Shigella Shigella spp. isolates (4 different spp.) P. aeruginosa 100 fg-10 fg/μL 7 Ureaplasma 1 pg/μL 14 Tested representative spp. strain of all serotypes of U. urealyticum and U. parvum (1-14) C. trachomatis 15 copies/μL 4 Tested representative isolates of serovars D, E, A. baumannii 10 fg-1 fg/μL 1 H, and F S. agalactiae 300 fg/μL 30 Tested representative (GBS) isolates from all serotypes (1A, 1B, 2-7) and non- typeable isolates (n = 2)

The target for real-time PCR detection of E. coli also reacts with the Shigellae. This E. coli/Shigella assay successfully detected all E. coli types tested, including representative isolates of each virotype (EHEC, EPEC, ETEC, EAEC, and EIEC), but did not amplify the closely related species E. albertii, E. hermannii, or E. fergusonii. This assay also detected all four Shigella species (S. flexneri, S. sonnei, S. dysenteriae, and S. boydii), with the exception of S. dysenteriae serotype I. During development and validation of this assay, sporadic amplification signal in NTC reactions was occasionally observed. This was determined to occur due to residual E. coli DNA present in the enzyme preparation from the manufacturers, which varied between production lots. Residual E. coli DNA in extraction reagents may also contribute to this phenomenon during testing of clinical specimens. Crossing threshold (Ct) values for this sporadic amplification were generally found to be >30. For this reason, a Ct cutoff value of 30 was implemented.

Extraction of TNA from blood and saline: Direct comparison of Ct values of TNA extracted from blood spiked with gram-positive bacteria (S. aureus) revealed that the average Ct value was approximately 5.5 cycles lower for TNA extracted after incubation with pre-lysis enzymes compared to identical preparations without this pre-treatment step (FIG. 1A). Incubation of spiked blood specimens with TE buffer alone did not result in lower Ct values, indicating that the observed improvement in Ct values was a result of the pre-lysis enzyme treatment instead of simply dilution and heating. Pre-treatment with lytic enzymes had no significant impact on Ct values of TNA from blood spiked with the gram-negative bacterium K. pneumoniae (FIG. 1B).

In addition, pre-lysis did not significantly impact target detection in saline spiked with the same serial dilutions. However, comparison of the Ct values for TNA extracted from blood and saline spiked with the same concentration of organisms revealed that detection in blood is significantly impaired relative to saline. The difference in Ct value for detection of the same number of bacteria in blood compared to saline ranged from 3.8 cycles at higher concentrations to 11.8 cycles at lower concentrations (mean difference in Ct value=6), including a complete lack of detection of the lowest concentration in blood (FIG. 1C).

TAC preparation: oligonucleotide concentration and assay replicates: The impact of oligonucleotide concentration on pathogen detection was briefly examined using the optimization TAC configuration. No significant difference in target amplification was observed at the limit of detection for any of the four targets examined. Additionally, testing of clinical specimens previously known to be positive for each of these pathogens did not reveal any oligonucleotide concentration-dependent difference in target detection.

Testing of serial dilutions of nucleic acid from M. pneumoniae, S. pneumoniae, S. agalactiae, and PIV2 revealed excellent concordance between replicates at higher TNA concentrations. In contrast, the number of replicate reactions in which amplification was observed decreased as the concentration approached the limit of detection for each assay. Testing five replicates allowed detection of less concentrated nucleic acid compared to two replicates. These results indicate that testing a higher number of replicates improves pathogen detection rates when the concentration of organisms is near the limit of detection.

Positive results in clinical specimens were confirmed by repeat testing in individual RT-qPCR reactions followed by confirmation of the appropriate size amplicon. The proportion of NP/OP and blood specimens identified as positive in more than half of assay replicates varied by target (FIG. 2). Overall, this proportion was significantly higher in NP/OP specimens (FIG. 2A) compared to whole blood (FIG. 2B). In other words, while the majority of NP/OP specimens that were positive were identified as positive in more than half of assay replicates, amplification of pathogen-specific targets in whole blood extracts occurred in a much smaller proportion of total replicates tested. Furthermore, average Ct values were higher in positive reactions containing TNA extracted from blood compared to NP/OP swabs. Together these data suggest that a higher number of assay replicates may identify pathogens which otherwise would be missed, particularly in whole blood specimens.

Enzyme system performance with TAC: Newer generations of enzyme mixes may result in improved detection of pathogen targets, particularly in the presence of molecules known to have inhibitory effects on real-time PCR, such as some blood components. The performance of two enzyme formulations, Ambion AGPATH-ID™ One-step RT-PCR kit (Applied Biosystems) and Quanta QSCRIPT™ XLT One-step RT-qPCR TOUGHMIX®, low ROX (Quanta Biosciences), at detecting targets in NP/OP specimens (n=18) and blood specimens (n=12) (FIG. 3) were compared. The median difference in Ct value for positive results in NP/OP specimens tested with TOUGHMIX® compared to AGPATH-ID™ enzyme varied by target (range 0-4), but overall improved Ct values were observed in reactions using TOUGHMIX® enzyme (FIG. 3A). An even more dramatic improvement in Ct values was observed for blood specimens tested with TOUGHMIX® compared to AGPATH-ID™; the median difference in Ct value ranged from 0.46 to 10.5 for various targets (FIG. 3B). In addition, additional positive results (n=16) for pathogen-specific targets were detected using the TOUGHMIX® enzyme mix that were not detected when testing the same specimen extract using AGPATH-ID™. This phenomenon was not limited to a single pathogen target, but rather occurred with 12 unique assays. While a few instances (n=5) were also observed where the reactions using AGPATH-ID™ yielded a positive result while the TOUGHMIX® reaction was negative, this occurred only when the Ct value with AGPATH-ID™ was >33, at the threshold where reproducibility between replicates is most commonly discordant. Overall, improved pathogen detection was observed using the TOUGHMIX® enzyme system, particularly in primary blood specimens.

EXAMPLE 4 Diagnostic Multiplex PCR Assay

This example describes exemplary methods that can be used to detect one or more of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli/Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcus agalactiae, and/or Neisseria meningitidis nucleic acids in a sample from a subject, thereby diagnosing the subject with infection with the detected organism(s). The methods can also be used to detect presence of one or more of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumooniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli/Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcus agalactiae, and/or Neisseria meningitidis in an environmental sample. One of ordinary skill in the art will appreciate that methods that deviate from these specific methods can also be used to successfully detect Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli/Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcus agalactiae, and/or Neisseria meningitidis nucleic acids in a sample.

Clinical samples are obtained from a subject (such as a subject suspected of having a CAP infection), such as a nasopharyngeal, oropharyngeal, or bronchial swab, bronchoalveolar lavage, or sputum, or an environmental sample is obtained, for example by swabbing a surface suspected of harboring one or more pathogens. DNA is extracted from the sample using routine methods (for example using a commercial kit).

Multiplex real-time PCR is performed in a reaction including a reaction mix (e.g., buffers, MgCl₂, dNTPs, and DNA polymerase), sample DNA (5 μl of nucleic acid extracted from the sample), and probes and primers (such as those in Table 1, above). The probes and primers are included in the reaction at concentrations of about 25 nM to 1 μM. The assay is performed using a real-time PCR system (such as the ABI 7500). Exemplary thermocycling conditions are 5 minutes at 95° C., followed by 45 cycles of 95° C. for 15 seconds and 60° C. for 1 minute. Positive samples are those with a positive C_(t) value for one or more pathogen probes.

EXAMPLE 5 Diagnostic Microfluidic Card Assay

This example describes exemplary methods that can be used to simultaneously detect one or more of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumooniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli/Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcus agalactiae, and/or Neisseria meningitidis nucleic acids in a sample from a subject, thereby diagnosing the subject with infection with the detected organism(s), or presence of the detected organism(s) in an environmental sample. One of ordinary skill in the art will appreciate that methods that deviate from these specific methods can also be used to successfully detect one or more of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumooniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli/Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcus agalactiae, and/or Neisseria meningitidis nucleic acids in a sample.

Clinical samples are obtained from a subject (such as a subject suspected of having a pathogenic infection), such as a nasopharyngeal, oropharyngeal, or bronchial swab, bronchoalveolar lavage, or sputum, or an environmental sample is obtained, for example by swabbing a surface suspected of harboring one or more pathogens. Nucleic acids (such as DNA, RNA, or total nucleic acid) are extracted from the sample using routine methods (for example using a commercial kit).

A microfluidic card (such as a TAQMAN® Array card (also known as a TAQMAN® Low Density Array card); Applied Biosystems, Foster City, Calif.) including primers and probes for one or more of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumooniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli/Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcus agalactiae, and/or Neisseria meningitidis is utilized. Individual wells of the card include primers and probe for a single pathogen, which are preloaded and dried onto the designated wells (for example in duplicate). The card may include at least one well containing Acinetobacter baumannii primers and probe (SEQ ID NOs: 12-14), at least one well containing Pseudomonas aeruginosa primers and probe (SEQ ID NOs: 15-17), at least one well containing Klebsiella pneumoniae primers and probe (SEQ ID NOs: 18-20 and/or SEQ ID NOs: 58-60), at least one well containing Toxoplasma gondii primers and probe (SEQ ID NOs: 21-23), at least one well containing Moraxella catarrhalis primers and probe (SEQ ID NOs: 24-26), at least one well containing E. coli/Shigella primers and probe (SEQ ID NOs: 27-29), at least one well containing Staphylococcus aureus primers and probe (SEQ ID NOs: 30-32), at least one well containing Pneumocystis jirovecii primers and probe (SEQ ID NOs: 33-35), at least one well containing Chlamydia trachomatis primers and probe (SEQ ID NOs: 36-38), at least one well containing Ureaplasma urealyticum primers and probe (SEQ ID NOs: 39-41), at least one well containing Ureaplasma parvum primers and probe (SEQ ID NOs: 42-44), at least one well containing Ureaplasma spp. primers and probe (SEQ ID NOs: 45-47), at least one well containing Bartonella spp. primers and probe (SEQ ID NOs: 48-50 or SEQ ID NOs: 49-51), at least one well containing Streptococcus agalactiae primers and probe (SEQ ID NOs: 54-56), or at least one well containing Neisseria meningitidis primers and probe (SEQ ID NOs: 62-64). Each probe includes a 5′ FAM fluorophore. Unless otherwise noted, each probe also includes a 3′ BHQ1 quencher. The P. jirovecii probe (SEQ ID NO: 35), the U. parvum probe (SEQ ID NO: 44), and the Ureaplasma spp. probe (SEQ ID NO: 47) each also include an internal BHQ1 quencher. One of ordinary skill in the art can select different labels and quenchers with only routine testing.

A master mix, including 1× RT-PCR buffer, 1× RT-PCR enzyme and nucleic acids isolated from a sample is applied to the microfluidic card utilizing the loading ports. The cards are centrifuged, sealed, and placed in a thermocycler (such as the Applied Biosystems VIIA™ 7 real-time PCR platform). Cycling conditions are 45° C. for 10 minutes, 94° for 10 minutes, and 45 cycles of 94° C. for 30 seconds and 60° C. for 1 minute (although these conditions can be adjusted by one of ordinary skill in the art to obtain optimal results, for example 55° C., instead of 60° C., in some examples). Positive samples are those with a positive C_(t) value for one or more probes.

In view of the many possible embodiments to which the principles of the disclosure may be applied, it should be recognized that the illustrated embodiments are only examples and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims. 

We claim:
 1. A method for detecting presence of Klebsiella pneumoniae in a sample, comprising: contacting the sample with a nucleic acid probe comprising the nucleic acid sequence of SEQ ID NO: 20 or SEQ ID NO: 60, or the reverse complement thereof, and a donor fluorophore, an acceptor fluorophore, or a combination thereof; amplifying a nucleic acid from Klebsiella pneumoniae, wherein the amplifying comprises contacting the sample with a pair of primers comprising the nucleic acid sequences of SEQ ID NOs: 18 and 19 or SEQ ID NOs: 58 and 59; and detecting hybridization between the nucleic acid probe and a nucleic acid in the sample, wherein detection of hybridization indicates the presence of Klebsiella pneumoniae in the sample.
 2. The method of claim 1, further comprising detecting one or more of Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Streptococcus agalactiae, and Bartonella spp. in the sample by: (a) contacting the sample with one or more nucleic acid probes, wherein: the pathogen is Pseudomonas aeruginosa and the probe comprises or consists of the nucleic acid sequence set forth as SEQ ID NO: 17; the pathogen is Bartonella spp. and the probe comprises or consists of the nucleic acid sequence set forth as SEQ ID NO: 50; the pathogen is Acinetobacter baumannii and the probe comprises or consists of the nucleic acid sequence set forth as SEQ ID NO: 14; the pathogen is Staphylococcus aureus and the probe comprises or consists of the nucleic acid sequence set forth as SEQ ID NO: 32; the pathogen is Toxoplasma gondii and the probe comprises or consists of the nucleic acid sequence set forth as SEQ ID NO: 23; the pathogen is Moraxella catarrhalis and the probe comprises or consists of the nucleic acid sequence set forth as SEQ ID NO: 26; the pathogen is Escherichia coli and/or Shigella and the probe comprises or consists of the nucleic acid sequence set forth as SEQ ID NO: 29; the pathogen is Chlamydia trachomatis and the probes comprises or consists of the nucleic acid sequence set forth as SEQ ID NO: 38; the pathogen is Ureaplasma urealyticum and the probe comprises or consists of the nucleic acid sequence set forth as SEQ ID NO: 41; the pathogen is Ureaplasma parvum and the probe comprises or consists of the nucleic acid sequence set forth as SEQ ID NO: 44; the pathogen is Ureaplasma spp. and the probe comprises or consists of the nucleic acid sequence set forth as SEQ ID NO: 47; and/or the pathogen is Streptococcus agalactiae and the probe comprises or consists of the nucleic acid sequence set forth as SEQ ID NO: 56; and (b) detecting hybridization between the one or more nucleic acid probes and a nucleic acid in the sample, wherein detection of hybridization in the sample indicates the presence of one or more of said pathogens in the sample.
 3. The method of claim 2, further comprising amplifying a nucleic acid from one or more of Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Streptococcus agalactiae, and Bartonella spp. by contacting the nucleic acid with at least one primer, wherein: the pathogen is Pseudomonas aeruginosa and the at least one primer comprises the nucleic acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 16; the pathogen is Bartonella spp. and the at least one primer comprises the nucleic acid sequence set forth in SEQ ID NO: 48, SEQ ID NO: 49, or SEQ ID NO: 51; the pathogen is Acinetobacter baumannii and the at least one primer comprises the nucleic acid sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 13; the pathogen is Staphylococcus aureus and the at least one primer comprises the nucleic acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 31; the pathogen is Toxoplasma gondii and the at least one primer comprises the nucleic acid sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 22; the pathogen is Moraxella catarrhalis and the at least one primer comprises the nucleic acid sequence set forth in SEQ ID NO: 24 or SEQ ID NO: 25; the pathogen is Escherichia coli and/or Shigella and the at least one primer comprises the nucleic acid sequence set forth in SEQ ID NO: 27 or SEQ ID NO: 28; the pathogen is Chlamydia trachomatis and the at least one primer comprises the nucleic acid sequence set forth in SEQ ID NO: 36 or SEQ ID NO: 37; the pathogen is Ureaplasma urealyticum and the at least one primer comprises the nucleic acid sequence set forth in SEQ ID NO: 39 or SEQ ID NO: 40; the pathogen is Ureaplasma parvum and the at least one primer comprises the nucleic acid sequence set forth in SEQ ID NO: 42 or SEQ ID NO: 43; the pathogen is Ureaplasma spp. and the at least one primer comprises the nucleic acid sequence set forth in SEQ ID NO: 45 or SEQ ID NO: 46; and/or the pathogen is Streptococcus agalactiae and the at least one primer comprises the nucleic acid sequence set forth in SEQ ID NO: 54 or SEQ ID NO:
 55. 4. The method of claim 1, wherein the sample comprises a biological sample or environmental sample.
 5. The method of claim 4, wherein the sample is a biological sample comprising tissue, blood, serum, cerebral spinal fluid, middle ear fluid, bronchoalveolar lavage, tracheal aspirate, sputum, nasopharyngeal aspirate, oropharyngeal aspirate, or saliva.
 6. The method of claim 4, wherein the sample is an environmental sample comprising a food sample, a water sample, or a surface swab.
 7. An isolated nucleic acid probe consisting of the nucleic acid sequence of SEQ ID NO: 20 or SEQ ID NO: 60 and a donor fluorophore, an acceptor fluorophore, or a combination thereof.
 8. A kit for detection of Klebsiella, comprising the isolated nucleic acid probe of claim 7 and a pair of primers comprising the nucleic acid sequences of SEQ ID NOs: 18 and 19 or SEQ ID NOs: 58 and
 59. 9. The kit of claim 8, further comprising one or more primers comprising the nucleic acid sequence of any one of SEQ ID NOs: 12, 13, 15, 16, 21, 22, 24, 25, 27, 28, 30, 31, 36, 37, 39, 40, 42, 43, 45, 46, 48, 49, 51, 54, and
 55. 10. The kit of claim 9, wherein the one or more primers consist of the nucleic acid sequence of any one of SEQ ID NOs: 12, 13, 15, 16, 21, 22, 24, 25, 27, 28, 30, 31, 36, 37, 39, 40, 42, 43, 45, 46, 48, 49, 51, 54, and
 55. 11. The kit of claim 8, further comprising one or more isolated nucleic acid probes up to 40 nucleotides in length, comprising the nucleic acid sequence of any one of SEQ ID NOs: 14, 17, 23, 26, 29, 32, 38, 41, 44, 47, 50, and 56, and a donor fluorophore, an acceptor fluorophore, or a combination thereof. 